Anti-ERBB3 antibodies

ABSTRACT

Monoclonal antibodies that bind and inhibit activation of epidermal growth factor receptor related member ErbB3/HER3 are disclosed. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation of ErbB3/HER3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/987,374, filed Jan. 4, 2016, now U.S. Pat. No. 9,598,498, which is adivisional of U.S. patent application Ser. No. 13/919,582, filed Jun.17, 2013, now U.S. Pat. No. 9,228,021, which is a divisional of U.S.patent application Ser. No. 13/082,852 filed Apr. 8, 2011, now U.S. Pat.No. 8,481,687, which claims priority to and the benefit of U.S.Provisional Patent Application No. 61/322,712, filed Apr. 9, 2010; theentire contents of each of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 2, 2017, isnamed AVO009DV2C1_SL.txt and is 232,747 bytes in size.

FIELD OF THE INVENTION

The field of the invention is molecular biology, immunology andoncology. More particularly, the field is humanized antibodies that bindhuman ErbB3/HER3.

BACKGROUND

HER3/c-ErbB3 (referred to herein as ErbB3) is a member of the epidermalgrowth factor receptor (EGFR) family. ErbB3 binds neuregulin/heregulin(NRG/HRG). Receptors in the EGFR family are single transmembranereceptors with an intracellular tyrosine kinase domain. While the otherEGFR family members, i.e., EGFR/HER1/ErbB1, HER2/ErbB2, and HER4/ErbB4,each have tyrosine kinase activity, ErbB3 has little or no tyrosinekinase activity, and thus is “kinase-dead.”

The extracellular domain (ECD) of the EGFR family contains four domains.Domains 1 and 3 (also known as domains L1 and L2) are responsible forligand binding. Cysteine-rich domains 2 and 4 (also known as domains C1and C2) are involved in dimerization with receptor partners. Upon ligandbinding, the ECD undergoes conformational changes. The interaction ofdomains 2 and 4, which maintains the tethered (inactive) conformation ofthe receptor, is relieved, and an extended (active) conformation isadopted. The extended conformation favors dimerization with otherreceptor partners. HER2/ErbB2 is the only exception to this generalrule, i.e., Her2-ECD is constitutively in the extended conformation. Noligand for HER2 has been identified thus far.

Because ErbB3 lacks an intrinsic kinase activity, it must dimerize withanother active tyrosine kinase receptor to be activated by tyrosinephosphorylation. Dimerization can occur between two different receptors(heterodimerization), e.g., ErbB3 and EGFR/HER1/ErbB1, HER2/ErbB2, orHER4/ErbB4. Recently, ErbB3 was also shown to dimerize with MET. Uponassociation with another tyrosine kinase receptor, ErbB3 is activated byphosphorylation of at least nine tyrosine residues in the ErbB3intracellular domain, and then rapidly associates with adaptors ordownstream signaling molecules. Six of the ErbB3 phosphorylated tyrosineresidues associate directly with the p85 subunit of Phosphatidylinositol3-Kinase (PIK3), which results in activation of the cellular survivalpathway controlled by the PI3K/Akt axis. Constitutive activation ofErbB3 by unregulated dimerization and/or unregulated phosphorylation ofErbB3 can lead to certain cancers.

Overexpression of ErbB3 is associated with poor prognosis in variouscarcinomas (e.g., breast, ovarian, prostate, colorectal, pancreatic,gastric, and head and neck cancers). Overexpression of ErbB3 alsocorrelates with local to distal metastasis in lung, gastric, andcolorectal cancers, and bone invasion in prostate cancer (Sithanandam etal., 2008, CANCER GENE THERAPY 15:413). Overexpression of ErbB3 has beenlinked to resistance to several cancer treatments, including treatmentwith EGFR tyrosine kinase inhibitors in non-small cell lung cancer(NSCLC) and head and neck cancers, treatment with Her2 inhibitor inbreast cancers, and treatment with radiotherapy in pancreatic cancers.Moreover, overexpression of NRG, a ligand for ErbB3, was also linked toresistance to EGFR tyrosine kinase inhibitor treatment. Chen et al.describe the use of anti-ErbB3 monoclonal antibodies that inhibit NRGfunction and show growth inhibitory activity against breast and ovariancancer cells (Chen et al., 1996, J. BIOL. CHEM. 271: 7620).

There is a need for improved anti-ErbB3 antibodies that can be used astherapeutic agents.

SUMMARY

The invention is based on the discovery of a family of antibodies thatspecifically bind human ErbB3. The antibodies contain ErbB3 bindingsites based on CDRs that specifically bind human ErbB3. When used astherapeutic agents, the antibodies are engineered, e.g., humanized, toreduce or eliminate an immune response when administered to a humanpatient.

The antibodies disclosed herein prevent or inhibit the activation ofhuman ErbB3. In some embodiments, the antibodies prevent ErbB3 frombinding to a ligand, e.g., NRG/HRG, thereby neutralizing the biologicalactivity of ErbB3. In other embodiments, the anti-ErbB3 antibodiesinhibit ErbB3 dimerization, thereby neutralizing the biological activityof ErbB3. The antibodies disclosed herein can be used to inhibit theproliferation of tumor cells in vitro or in vivo. When administered to ahuman cancer patient (or an animal model such as a mouse model), theantibodies inhibit or reduce tumor growth in the human patient (oranimal model).

These and other aspects and advantages of the invention are illustratedby the following figures, detailed description and claims. As usedherein, “including” means without limitation, and examples cited arenon-limiting.

DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1 (prior art) is a schematic representation of a typical antibody.

FIG. 2 is a schematic diagram showing the amino acid sequence of thecomplete immunoglobulin heavy chain variable region of the antibodiesdenoted as 04D01, 09D03, 11G01, 12A07, 18H02, 22A02, and 24C05. Theamino acid sequences for each antibody are aligned against one another,and Complementary Determining Sequences (CDR) (Kabat definition), CDR₁,CDR₂, and CDR₃, are identified in boxes. The unboxed sequences representframework (FR) sequences.

FIG. 3 is a schematic diagram showing the CDR₁, CDR₂, and CDR₃ sequences(Kabat definition) for each of the immunoglobulin heavy chain variableregion sequences in FIG. 2.

FIG. 4 is a schematic diagram showing the amino acid sequence of thecomplete immunoglobulin light chain variable region of antibodies 04D01,09D03, 11G01, 12A07, 18H02, 22A02, and 24C05. The amino acid sequencesfor each antibody are aligned against one another, and CDR₁, CDR₂, andCDR₃ sequences (Kabat definition) are identified in boxes. The unboxedsequences represent framework (FR) sequences.

FIG. 5 is a schematic diagram showing the CDR₁, CDR₂, and CDR₃ sequences(Kabat definition) for each of the immunoglobulin light chain variableregion sequences in FIG. 4.

FIGS. 6A and 6B are graphs summarizing results from an experiment tomeasure the neutralization activity of negative control (murine IgG (Δ))and anti-ErbB3 monoclonal antibodies 04D01 (▪), 12A07 (◯), 18H02 (●),22A02 (□) and 24C05 (Q) to inhibit NRG1-β1 binding to hErbB3 (FIG. 6A)and to measure the enhanced binding of NRG1-β1 to rhErbB3 by theanti-ErbB3 mAb 09D03 (▴) and 11G01 (*) (FIG. 6B).

FIG. 7 is a graph summarizing results from an experiment to measure theneutralization activity of negative control (murine IgG) and anti-ErbB3monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05to inhibit NRG1-al binding to rhErbB3.

FIG. 8 is a graph summarizing results from an experiment to measure thecell surface recognition of the anti-ErbB3 antibodies of the chimericprotein Her2/3d2 expressed at the surface of CHO cells.

FIG. 9 is a graph summarizing results from an experiment to measure theanti-proliferation activity of negative control IgG (murine IgG (Δ)) andanti-ErbB3 monoclonal antibodies 04D01 (▪), 09D03 (▾), 11G01 (♦), 12A07(◯), 18H02 (⋄), 22A02 (●) and 24C05 (□) in BaF/3 cells expressing Her2and ErbB3 in presence of NRG1-β1.

FIG. 10 is a graph summarizing results from an experiment to measure theanti-proliferation activity of anti-ErbB3 monoclonal antibodies 04D01(▪), 09D03 (▾), 11G01 (♦), 12A07 (▪), 18H02 (⋄), 22A02 (●) and 24C05 (□)in MCF7 cells in the presence of NRG1-β1.

FIG. 11 is a graph summarizing results from an experiment to measure theanti-proliferation activity of negative control (murine IgG) andanti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02,22A02 and 24C05 in SKBR-3 cells treated with 5 μg/ml of antibodies inthe presence of serum.

FIG. 12 is graph summarizing results from an experiment to measure theinhibitory activity of negative control IgG and anti-ErbB3 monoclonalantibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 on thephosphorylation of ErbB3 induced by NRG in SKBR-3 cells. No antibody/noligand and no antibody controls are also shown.

FIGS. 13A and 13B are graphs representing results from an experiment tomeasure the inhibitory activity of anti-ErbB3 monoclonal antibodies04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 on thephosphorylation of Akt in response to NRG1-31 in MCF7 cells (FIG. 13A)and in DU145 cells (FIG. 13B) as determined by ELISA. No antibody/noligand and no antibody controls are also shown.

FIG. 14 is a graph summarizing results from an experiment to measure thetumor inhibitory activity of the anti-ErbB3 antibodies 04D01 (Δ), 09D03(*), 11G01 (□), 12A07 (▴), 18H02 (●), 22A02 (▪), 24C05 (◯) and a humanIgG control (- -▪- -) dosed at 20 mg/kg in a BxPC3 pancreatic tumorxenograft model in CB 17 SCID mice (vehicle control, PBS (♦)).

FIG. 15 is a schematic diagram showing the amino acid sequences of thecomplete heavy chain variable region of 24C05 and the complete humanizedheavy chain variable regions denoted as Sh24C05 Hv3-7, Sh24C05 Hv3-11,Sh24C05 Hv3-11 N62S, Sh24C05 Hv3-21, Sh24C05 Hv3-23, Sh24C05 Hv3-30, andHu24C05 HvA. The amino acid sequences for each heavy chain variableregions are aligned against one another, and Complementary DeterminingSequences (CDR) (Kabat definition), CDR₁, CDR₂, and CDR₃, are identifiedin boxes. The unboxed sequences represent framework (FR) sequences.

FIG. 16 is a schematic diagram showing the amino acid sequences of thecomplete light chain variable region of 24C05 and the complete humanizedlight chain variable regions denoted as Sh24C05 Kv1-9, Sh24C05 Kv1-16,Sh24C05 Kv1-17, Sh24C05 Kv1-33, Sh24C05 Kv1-39, and Hu24C05 KvA. Theamino acid sequences for each light chain variable regions are alignedagainst one another, and CDR₁, CDR₂, and CDR₃ sequences (Kabatdefinition) are identified in boxes. The unboxed sequences representframework (FR) sequences.

FIG. 17 are BIACORE® sensorgrams representing results from an experimentto measure the kinetic values of anti-ErbB3 monoclonal antibodies,Sh24C05-31 N62S-IgG1, Ab#6, U1-53, and U1-59.

FIG. 18A is a graph summarizing results from an experiment to measurethe neutralization activity of negative control (human IgG (□)) andanti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1 (▴), Sh24C05-25N62S-IgG2 (Δ), Sh24C05-31 N62S-IgG1 (●) and Sh24C05-31 N62S-IgG2 (◯).FIG. 18B is a graph summarizing results from an experiment to measurethe neutralization activity of human IgG (□) and anti-ErbB3 monoclonalantibodies Ab#6 IgG2 (▾), U1-53 (⋄) and U1-59 (▪).

FIG. 19A is a graph summarizing results from an experiment to measurethe inhibitory activity of negative control (human IgG (□)) andanti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1 (▴), Sh24C05-25N62S-IgG2 (Δ), Sh24C05-31 N62S-IgG1 (●) and Sh24C05-31 N62S-IgG2 (◯) inBaF/3 cells expressing Her2 and ErbB3 in the presence of NRG1-β1. FIG.19B is a graph summarizing results from an experiment to measure theinhibitory activity of human IgG (□) and anti-ErbB3 monoclonalantibodies Sh24C05-31 N62S-IgG1 (●), Ab#6 IgG2 (∇), U1-53 (♦) and U1-59(▪) in BaF/3 cells expressing Her2 and ErbB3 in the presence of NRG1-β1.

FIG. 20 is a graph summarizing results from an experiment to measure theinhibitory activity of negative control (human IgG) and anti-ErbB3monoclonal antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2,Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 on the steady statephosphorylation of ErbB3 in growing SKBR-3 cells.

FIG. 21 is a graph summarizing results from an experiment to measure thedegradation of ErbB3 receptor by negative control (human IgG) andanti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 in growingSKBR-3 cells.

FIG. 22 is a graph summarizing the results from an experiment to measuretumor inhibitory activity of a human IgG, murine IgG, or anti-ErbB3monoclonal antibodies dosed at 2 mg/kg in a BxPC3 pancreatic tumorxenograft model in CB 17 SCID mice (murine 24C05 (Δ), Sh24C05-31 N62SIgG1 (●), Sh24C05-31 N62S IgG2 (♦), Sh24C05-25 N62S IgG1 (▴), Sh24C05-25N62S IgG2 (▪), vehicle control (□), murine IgG (x), and human IgG (⋄)).

FIG. 23A is a graph summarizing the results from an experiment tomeasure tumor inhibitory activity of a murine IgG or anti-ErbB3monoclonal antibodies dosed at 5 mg/kg in a Calu-3 non-small cell lungcancer xenograft model in NCR nude mice (vehicle control (□), murine IgG(x), Sh24C05-31 N62S IgG1 (▴), Ab#6 IgG2 (●), and U1-59 (▪)).

FIG. 23B is a graph summarizing the results from an experiment tomeasure tumor inhibitory activity of a murine IgG or anti-ErbB3monoclonal antibodies dosed at 10 mg/kg in a Calu-3 non-small cell lungcancer xenograft model in NCR nude mice (vehicle control (□), murine IgG(x), Sh24C05-31 N62S IgG1 (▴), Ab#6 IgG2 (●), and U1-59 (▪)).

FIG. 23C is a graph summarizing the results from an experiment tomeasure tumor inhibitory activity of a murine IgG or anti-ErbB3monoclonal antibodies dosed at 20 mg/kg in a Calu-3 non-small cell lungcancer xenograft model in NCR nude mice (vehicle control (□), murine IgG(x), Sh24C05-31 N62S IgG1 (▴), Ab#6 IgG2 (●), and U1-59 (▪)).

FIG. 24 is a graph summarizing the results from an experiment to measuretumor inhibitory activity of a human IgG, murine or anti-ErbB3monoclonal antibodies in a MDA-MB-453 breast cancer xenograft model inNOD SCID mice (vehicle control (□), human IgG (x), Sh24C05-31 N62S IgG1dosed at 5 mg/kg (⋄), Sh24C05-31 N62S IgG1 dosed at 10 mg/kg (Δ),Sh24C05-31 N62S IgG1 dosed at 20 mg/kg (▴), Ab#6 IgG2 dosed at 10 mg/kg(●), and U1-59 dosed at 10 mg/kg (▪)).

DETAILED DESCRIPTION

The ErbB3 antibodies disclosed herein are based on the antigen bindingsites of certain monoclonal antibodies selected for their ability toneutralize the biological activity of human ErbB3 polypeptides. Theantibodies contain immunoglobulin variable region CDR sequences thatdefine a binding site for ErbB3. In some embodiments, the antibodiesprevent ErbB3 from binding to a ligand, e.g., NRG/HRG, therebyneutralizing the biological activity of ErbB3. In other embodiments, theanti-ErbB3 antibodies inhibit ErbB3 dimerization, thereby neutralizingthe biological activity of ErbB3. In still other embodiments, theanti-ErbB3 antibodies inhibit phosphorylation of ErbB3 and downstreamsignaling.

Because of the neutralizing activity of these antibodies, they areuseful for inhibiting the growth and/or proliferation of certain cancercells and tumors. The antibodies can be engineered to minimize oreliminate an immune response when administered to a human patient. Insome embodiments, the antibodies are fused or conjugated to othermoieties, such as detectable labels or effector molecules such as smallmolecule toxins.

I. Antibodies that Bind ErbB3

In some embodiments, the antibody comprises: (a) an immunoglobulin heavychain variable region comprising the structureCDR_(H1)-CDR_(H2)-CDR_(H3) and (b) immunoglobulin light chain variableregion, wherein the heavy chain variable region and the light chainvariable region together define a single binding site for binding humanErbB3. A CDR_(H1) comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 5 (04D01), SEQ ID NO: 15 (09D03), SEQ IDNO: 25 (11G01), SEQ ID NO: 34 (12A07), SEQ ID NO: 41 (18H02), SEQ ID NO:51 (22A02), SEQ ID NO: 57 (24C05), and SEQ ID NO: 75 (24C05); a CDR_(H2)comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 6 (04D01), SEQ ID NO:16 (09D03), SEQ ID NO: 26 (11G01), SEQID NO: 35 (12A07), SEQ ID NO: 42 (18H02), SEQ ID NO: 52 (22A02), SEQ IDNO: 58 (24C05), and SEQ ID NO: 148 (Sh24C05 Hv3-11 N62S); and a CDR_(H3)comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 7 (04D01), SEQ ID NO: 17 (09D03), SEQ ID NO: 27 (11G01), SEQID NO: 36 (12A07, 22A02), SEQ ID NO: 43 (18H02), and SEQ ID NO: 59(24C05). Throughout the specification a particular SEQ ID NO. isfollowed in parentheses by the antibody that was the origin of thatsequence. For example, “SEQ ID NO: 5 (04D01)” means that SEQ ID NO: 5comes from antibody 04D01.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 5 (04D01), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 6 (04D01), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 7 (04D01).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 15 (09D03), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 16 (09D03), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 17 (09D03).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 25 (11601), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 26 (11601), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 27 (11601).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 34 (12A07), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 35 (12A07), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 36 (12A07, 22A02).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 41 (18H02), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 42 (18H02), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 43 (18H02).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 51 (22A02), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 52 (22A02), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 36 (12A07, 22A02).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 57 (24C05) or SEQ ID NO: 75 (24C05), a CDR_(H2)comprising the amino acid sequence of SEQ ID NO: 58 (24C05), and aCDR_(H3) comprising the amino acid sequence of SEQ ID NO: 59 (24C05).

In certain embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 57 (24C05), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 58 (24C05), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 59 (24C05).

In other embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising the amino acidsequence of SEQ ID NO: 75 (24C05), a CDR_(H2) comprising the amino acidsequence of SEQ ID NO: 58 (24C05), and a CDR_(H3) comprising the aminoacid sequence of SEQ ID NO: 59 (24C05).

In certain embodiments, the antibody comprises an immunoglobulin heavychain variable region a CDR_(H1) comprising the amino acid sequence ofSEQ ID NO: 57 (24C05) or SEQ ID NO: 75 (24C05), a CDR_(H2) comprisingthe amino acid sequence of SEQ ID NO: 148 (Sh24C05 Hv3-11 N62S), and aCDR_(H3) comprising the amino acid sequence of SEQ ID NO: 59 (24C05).

Preferably, the CDR_(H1), CDR_(H2), and CDR_(H3) sequences areinterposed between human or humanized immunoglobulin FRs. The antibodycan be an intact antibody or an antigen-binding antibody fragment.

In some embodiments, the antibody comprises (a) an immunoglobulin lightchain variable region comprising the structureCDR_(L1)-CDR_(L2)-CDR_(L3), and (b) an immunoglobulin heavy chainvariable region, wherein the IgG light chain variable region and the IgGheavy chain variable region together define a single binding site forbinding human ErbB3. A CDR_(L1) comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 8 (04D01, 12A07,22A02), SEQ ID NO: 18 (09D03), SEQ ID NO: 28 (11G01), SEQ ID NO: 44(18H02), and SEQ ID NO: 60 (24C05); a CDR_(L2) comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 9 (04D01,11G01, 12A07, 22A02), SEQ ID NO: 19 (09D03), SEQ ID NO: 45 (18H02), andSEQ ID NO: 61 (24C05); and a CDR_(L3) comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 10 (04D01, 12A07,22A02), SEQ ID NO: 20 (09D03), SEQ ID NO: 29 (11G01), SEQ ID NO: 46(18H02), and SEQ ID NO: 62 (24C05).

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising: a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 8 (04D01, 12A07, 22A02); a CDR_(L2) comprisingthe amino acid sequence of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02);and a CDR_(L3) comprising the amino acid sequence of SEQ ID NO: 10(04D01, 12A07, 22A02).

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising: a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 18 (09D03); a CDR_(L2) comprising the amino acidsequence of SEQ ID NO: 19 (09D03); and a CDR_(L3) comprising the aminoacid sequence of SEQ ID NO: 20 (09D03).

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising: a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 28 (11G01); a CDR_(L2) comprising the amino acidsequence of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02); and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO: 29 (11G01).

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising: a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 44 (18H02); a CDR_(L2) comprising the amino acidsequence of SEQ ID NO: 45 (18H02); and a CDR_(L3) comprising the aminoacid sequence of SEQ ID NO: 46 (18H02).

In one embodiment, the antibody comprises an immunoglobulin light chainvariable region comprising: a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 60 (24C05); a CDR_(L2) comprising the amino acidsequence of SEQ ID NO: 61 (24C05); and a CDR_(L3) comprising the aminoacid sequence of SEQ ID NO: 62 (24C05).

Preferably, the CDR_(L1), CDR_(L2), and CDR_(L3) sequences areinterposed between human or humanized immunoglobulin FRs. The antibodycan be an intact antibody or an antigen-binding antibody fragment.

In some embodiments, the antibody comprises: (a) an IgG heavy chainvariable region comprising the structure CDR_(H1)-CDR_(H2)-CDR_(H3) and(b) an IgG light chain variable region comprising the structureCDR_(L1)-CDR_(L2)-CDR_(L3), wherein the heavy chain variable region andthe light chain variable region together define a single binding sitefor binding human ErbB3. The CDR_(H1) is an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 5 (04D01), SEQ ID NO:15 (09D03),SEQ ID NO: 25 (11G01), SEQ ID NO: 34 (12A07), SEQ ID NO: 41 (18H02), SEQID NO: 51 (22A02), SEQ ID NO: 57 (24C05), and SEQ ID NO: 75 (24C05); theCDR_(H2) is an amino acid sequence selected from the group consisting ofSEQ ID NO: 6 (04D01), SEQ ID NO:16 (09D03), SEQ ID NO: 26 (11G01), SEQID NO: 35 (12A07), SEQ ID NO: 42 (18H02), SEQ ID NO: 52 (22A02), SEQ IDNO: 58 (24C05), and SEQ ID NO: 148 (Sh24C05 Hv3-11 N62S); and theCDR_(H3) is an amino acid sequence selected from the group consisting ofSEQ ID NO: 7 (04D01), SEQ ID NO: 17 (09D03), SEQ ID NO: 27 (11G01), SEQID NO: 36 (12A07, 22A02), SEQ ID NO: 43 (18H02), and SEQ ID NO: 59(24C05). The CDR_(L1) is an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8 (04D01, 12A07, 22A02), SEQ ID NO: 18 (09D03),SEQ ID NO: 28 (11G01), SEQ ID NO: 44 (18H02), and SEQ ID NO: 60 (24C05);the CDR_(L2) is an amino acid sequence selected from the groupconsisting of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02), SEQ ID NO: 19(09D03), SEQ ID NO: 45 (18H02), and SEQ ID NO: 61 (24C05); and theCDR_(L3) is an amino acid sequence selected from the group consisting ofSEQ ID NO: 10 (04D01, 12A07, 22A02), SEQ ID NO: 20 (09D03), SEQ ID NO:29 (11G01), SEQ ID NO: 46 (18H02), and SEQ ID NO: 62 (24C05).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region selected from the group consisting of SEQ ID NO: 2(04D01), SEQ ID NO: 12 (09D03), SEQ ID NO: 22 (11G01), SEQ ID NO: 31(12A07), SEQ ID NO: 38 (18H02), SEQ ID NO: 48 (22A02), SEQ ID NO: 54(24C05), and SEQ ID NO: 154 (Sh24C05 Hv3-11 N62S), and an immunoglobulinlight chain variable region selected from the group consisting of SEQ IDNO: 4 (04D01), SEQ ID NO: 14 (09D03), SEQ ID NO: 24 (11G01), SEQ ID NO:33 (12A07), SEQ ID NO: 40 (18H02), SEQ ID NO: 50 (22A02), SEQ ID NO: 56(24C05), SEQ ID NO: 166 (Sh24C05 Kv1-16), and SEQ ID NO: 168 (Sh24C05Kv1-17).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO: 2(04D01), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 4 (04D01).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:12 (09D03), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 14 (09D03).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:22 (11G01), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 24 (11G01).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:31 (12A07), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 33 (12A07).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:38 (18H02), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 40 (18H02).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:48 (22A02), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 50 (22A02).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:54 (24C05), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 56 (24C05).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:154 (Sh24C05 Hv3-11 N62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO: 166 (Sh24C05Kv1-16).

In another embodiment, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:154 (Sh24C05 Hv3-11 N62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO: 168 (Sh24C05Kv1-17).

In other embodiments, the antibody comprises (i) an immunoglobulin heavychain selected from the group consisting of SEQ ID NO: 109 (04D01), SEQID NO: 113 (09D03), SEQ ID NO: 117 (11G01), SEQ ID NO: 121 (12A07), SEQID NO: 125 (18H02), SEQ ID NO: 129 (22A07), SEQ ID NO: 133 (24C05), SEQID NO: 190 (Sh24C05 Hv3-11 N62S IgG1), and SEQ ID NO: 192 (Sh24C05Hv3-11 N62S IgG2), and (ii) an immunoglobulin light chain selected fromthe group consisting of SEQ ID NO: 111 (04D01), SEQ ID NO: 115 (09D03),SEQ ID NO: 119 (11G01), SEQ ID NO: 123 (12A07), SEQ ID NO: 127 (18H02),SEQ ID NO: 131 (22A07), SEQ ID NO: 135 (24C05), SEQ ID NO: 204 (Sh24C05Kv1-16 kappa), and SEQ ID NO: 206 (Sh24C05 Kv1-17 kappa).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 109 (04D01), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 111 (04D01).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 113 (09D03), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 115 (09D03).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 117 (11G01), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 119 (11G01).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 121 (12A07), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 123 (12A07).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 125 (18H02), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 127 (18H02).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 129 (22A02), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 131 (22A02).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 133 (24C05), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 135 (24C05).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 190 (Sh24C05Hv3-11 N62S IgG1), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 204 (Sh24C05 Kv1-16 kappa).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 192 (Sh24C05Hv3-11 N62S IgG2), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 204 (Sh24C05 Kv1-16 kappa).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 190 (Sh24C05Hv3-11 N62S IgG1), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 206 (Sh24C05 Kv1-17 kappa).

In another embodiment, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 192 (Sh24C05Hv3-11 N62S IgG2), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 206 (Sh24C05 Kv1-17 kappa).

As used herein, unless otherwise indicated, the term “antibody” means anintact antibody (e.g., an intact monoclonal antibody) or antigen-bindingfragment of a antibody (e.g., an antigen-binding fragment of amonoclonal antibody), including an intact antibody or antigen-bindingfragment that has been modified, engineered, or chemically conjugated.Examples of antibodies that have been modified or engineered includechimeric antibodies, humanized antibodies, and multispecific antibodies(e.g., bispecific antibodies). Examples of antigen-binding fragmentsinclude Fab, Fab′, (Fab′)₂, Fv, single chain antibodies (e.g., scFv),minibodies, and diabodies. An example of a chemically conjugatedantibody is an antibody conjugated to a toxin moiety.

FIG. 1 shows a schematic representation of an intact monoclonal antibodythat contains four polypeptide chains. Two of the polypeptide chains arecalled immunoglobulin heavy chains (H chains), and two of thepolypeptide chains are called immunoglobulin light chains (L chains).The immunoglobulin heavy and light chains are connected by an interchaindisulfide bond. The immunoglobulin heavy chains are connected byinterchain disulfide bonds. A light chain consists of one variableregion (V_(L) in FIG. 1) and one constant region (C_(L) in FIG. 1). Theheavy chain consists of one variable region (V_(H) in FIG. 1) and atleast three constant regions (CH₁, CH₂ and CH₃ in FIG. 1). The variableregions determine the specificity of the antibody.

Each variable region contains three hypervariable regions known ascomplementarity determining regions (CDRs) flanked by four relativelyconserved regions known as framework regions (FRs). The three CDRs,referred to as CDR₁, CDR₂, and CDR₃, contribute to the antibody bindingspecificity.

In certain embodiments, an isolated antibody that binds human ErbB3comprises an immunoglobulin heavy chain variable region comprising anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identical to the entire variable region or the framework regionsequence of SEQ ID NO: 2 (04D01), SEQ ID NO: 12 (09D03), SEQ ID NO: 22(11G01), SEQ ID NO: 31 (12A07), SEQ ID NO: 38 (18H02), SEQ ID NO: 48(22A02), SEQ ID NO: 54 (24C05), and SEQ ID NO: 154 (Sh24C05 Hv3-11N62S).

In certain embodiments, an isolated antibody that binds human ErbB3comprises an immunoglobulin light chain variable region comprising anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identical to the entire variable region or the framework regionsequence of SEQ ID NO: 4 (04D01), SEQ ID NO: 14 (09D03), SEQ ID NO: 24(11G01), SEQ ID NO: 33 (12A07), SEQ ID NO: 40 (18H02), SEQ ID NO: 50(22A02), SEQ ID NO: 56 (24C05), SEQ ID NO: 166 (Sh24C05 Kv1-16), and SEQID NO: 168 (Sh24C05 Kv1-17).

In each of the foregoing embodiments, it is contemplated herein thatimmunoglobulin heavy chain variable region sequences and/or light chainvariable region sequences that together bind human ErbB3 may containamino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acidsubstitutions, deletions, or additions) in the framework regions of theheavy and/or light chain variable regions.

In some embodiments, an isolated antibody binds hErbB3 with a K_(D) of350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 20 pM, 10pM or lower. Unless otherwise specified, K_(D) values are determined bysurface plasmon resonance methods. The surface plasmon resonance methodscan be performed using the conditions described, for example, inExamples 3 and 12, where the measurements were performed at 25° C. and37° C., respectively.

In some embodiments, the antibodies inhibit hErbB3 binding to NRG1-β1.For example, the antibodies can have an IC₅₀ (concentration at 50% ofmaximum inhibition) of about 5 nM, 2 nM or lower, when assayed using theprotocols described in Examples 4 and 13.

II. Production of Antibodies

Methods for producing antibodies disclosed herein are known in the art.For example, DNA molecules encoding light chain variable regions andheavy chain variable regions can be chemically synthesized using thesequence information provided herein. Synthetic DNA molecules can beligated to other appropriate nucleotide sequences, including, e.g.,constant region coding sequences, and expression control sequences, toproduce conventional gene expression constructs encoding the desiredantibodies. Production of defined gene constructs is within routineskill in the art. Alternatively, the sequences provided herein can becloned out of hybridomas by conventional hybridization techniques orpolymerase chain reaction (PCR) techniques, using synthetic nucleic acidprobes whose sequences are based on sequence information providedherein, or prior art sequence information regarding genes encoding theheavy and light chains of murine antibodies in hybridoma cells.

Nucleic acids encoding the antibodies disclosed herein can beincorporated (ligated) into expression vectors, which can be introducedinto host cells through conventional transfection or transformationtechniques. Exemplary host cells are E. coli cells, Chinese hamsterovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),and myeloma cells that do not otherwise produce IgG protein. Transformedhost cells can be grown under conditions that permit the host cells toexpress the genes that encode the immunoglobulin light and/or heavychain variable regions.

Specific expression and purification conditions will vary depending uponthe expression system employed. For example, if a gene is to beexpressed in E. coli, it is first cloned into an expression vector bypositioning the engineered gene downstream from a suitable bacterialpromoter, e.g., Trp or Tac, and a prokaryotic signal sequence. Theexpressed secreted protein accumulates in refractile or inclusionbodies, and can be harvested after disruption of the cells by Frenchpress or sonication. The refractile bodies then are solubilized, and theproteins refolded and cleaved by methods known in the art.

If a DNA construct encoding an antibody disclosed herein is to beexpressed in eukayotic host cells, e.g., CHO cells, it is first insertedinto an expression vector containing a suitable eukaryotic promoter, asecretion signal, IgG enhancers, and various introns. This expressionvector optionally contains sequences encoding all or part of a constantregion, enabling an entire, or a part of, a heavy and/or light chain tobe expressed. In some embodiments, a single expression vector containsboth heavy and light chain variable regions to be expressed.

The gene construct can be introduced into eukaryotic host cells usingconventional techniques. The host cells express V_(L) or V_(H)fragments, V_(L)-V_(H) heterodimers, V_(H)-V_(L) or V_(L)-V_(H) singlechain polypeptides, complete heavy or light immunoglobulin chains, orportions thereof, each of which may be attached to a moiety havinganother function (e.g., cytotoxicity). In some embodiments, a host cellis transfected with a single vector expressing a polypeptide expressingan entire, or part of, a heavy chain (e.g., a heavy chain variableregion) or a light chain (e.g., a light chain variable region). In otherembodiments, a host cell is transfected with a single vector encoding(a) a polypeptide comprising a heavy chain variable region and apolypeptide comprising a light chain variable region, or (b) an entireimmunoglobulin heavy chain and an entire immunoglobulin light chain. Instill other embodiments, a host cell is co-transfected with more thanone expression vector (e.g., one expression vector expressing apolypeptide comprising an entire, or part of, a heavy chain or heavychain variable region, and another expression vector expressing apolypeptide comprising an entire, or part of, a light chain or lightchain variable region).

A method of producing a polypeptide comprising an immunoglobulin heavychain variable region or a polypeptide comprising an immunoglobulinlight chain variable region may comprise growing a host cell transfectedwith an expression vector under conditions that permits expression ofthe polypeptide comprising the immunoglobulin heavy chain variableregion or the polypeptide comprising the immunoglobulin light chainvariable region. The polypeptide comprising a heavy chain variableregion or the polypeptide comprising the light chain variable regionthen may be purified using techniques well known in the art, e.g.,affinity tags such as glutathione-S-transferase (GST) and histidinetags.

A method of producing a monoclonal antibody that binds human ErbB3, oran antigen-binding fragment of the antibody, may comprise growing a hostcell transfected with: (a) an expression vector that encodes a completeor partial immunoglobulin heavy chain, and a separate expression vectorthat encodes a complete or partial immunoglobulin light chain; or (b) asingle expression vector that encodes both chains (e.g., complete orpartial chains), under conditions that permit expression of both chains.The intact antibody (or antigen-binding fragment) can be harvested andpurified using techniques well known in the art, e.g., Protein A,Protein G, affinity tags such as glutathione-S-transferase (GST) andhistidine tags. It is within ordinary skill in the art to express theheavy chain and the light chain from a single expression vector or fromtwo separate expression vectors.

III. Modifications to the Antibodies

Methods for reducing or eliminating the antigenicity of antibodies andantibody fragments are known in the art. When the antibodies are to beadministered to a human, the antibodies preferably are “humanized” toreduce or eliminate antigenicity in humans. Preferably, a humanizedantibody has the same or substantially the same affinity for the antigenas the non-humanized mouse antibody from which it was derived.

In one humanization approach, chimeric proteins are created in whichmouse immunoglobulin constant regions are replaced with humanimmunoglobulin constant regions. See, e.g., Morrison et al., 1984, PROC.NAT. ACAD. SCI. 81:6851-6855, Neuberger et al., 1984, NATURE312:604-608; U.S. Pat. No. 6,893,625 (Robinson); U.S. Pat. No. 5,500,362(Robinson); and U.S. Pat. No. 4,816,567 (Cabilly).

In an approach known as CDR grafting, the CDRs of the light and heavychain variable regions are grafted into frameworks from another species.For example, murine CDRs can be grafted into human FRs. In someembodiments, the CDRs of the light and heavy chain variable regions ofan anti-ErbB3 antibody are grafted onto human FRs or consensus humanFRs. To create consensus human FRs, FRs from several human heavy chainor light chain amino acid sequences are aligned to identify a consensusamino acid sequence. CDR grafting is described in U.S. Pat. No.7,022,500 (Queen); U.S. Pat. No. 6,982,321 (Winter); U.S. Pat. No.6,180,370 (Queen); U.S. Pat. No. 6,054,297 (Carter); U.S. Pat. No.5,693,762 (Queen); U.S. Pat. No. 5,859,205 (Adair); U.S. Pat. No.5,693,761 (Queen); U.S. Pat. No. 5,565,332 (Hoogenboom); U.S. Pat. No.5,585,089 (Queen); U.S. Pat. No. 5,530,101 (Queen); Jones et al. (1986)NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327;Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBSLETT 430: 92-94.

In an approach called “SUPERHUMANIZATION™,” human CDR sequences arechosen from human germline genes, based on the structural similarity ofthe human CDRs to those of the mouse antibody to be humanized. See,e.g., U.S. Pat. No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL169:1119-1125.

Other methods to reduce immunogenicity include “reshaping,”“hyperchimerization,” and “veneering/resurfacing.” See, e.g., Vaswami etal., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al.,1996, PROT. ENGINEER 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman).In the veneering/resurfacing approach, the surface accessible amino acidresidues in the murine antibody are replaced by amino acid residues morefrequently found at the same positions in a human antibody. This type ofantibody resurfacing is described, e.g., in U.S. Pat. No. 5,639,641(Pedersen).

Another approach for converting a mouse antibody into a form suitablefor medical use in humans is known as ACTIVMAB™ technology (Vaccinex,Inc., Rochester, N.Y.), which involves a vaccinia virus-based vector toexpress antibodies in mammalian cells. High levels of combinatorialdiversity of IgG heavy and light chains are said to be produced. See,e.g., U.S. Pat. No. 6,706,477 (Zauderer); U.S. Pat. No. 6,800,442(Zauderer); and U.S. Pat. No. 6,872,518 (Zauderer).

Another approach for converting a mouse antibody into a form suitablefor use in humans is technology practiced commercially by KaloBiosPharmaceuticals, Inc. (Palo Alto, Calif.). This technology involves theuse of a proprietary human “acceptor” library to produce an “epitopefocused” library for antibody selection.

Another approach for modifying a mouse antibody into a form suitable formedical use in humans is HUMAN ENGINEERING™ technology, which ispracticed commercially by XOMA (US) LLC. See, e.g., PCT Publication No.WO 93/11794 and U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and5,869,619.

Any suitable approach, including any of the above approaches, can beused to reduce or eliminate human immunogenicity of an antibodydisclosed herein.

Methods of making multispecific antibodies are known in the art.Multi-specific antibodies include bispecific antibodies. Bispecificantibodies are antibodies that have binding specificities for at leasttwo different epitopes. Exemplary bispecific antibodies bind to twodifferent epitopes of the antigen of interest. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.,F(ab′)₂ bispecific antibodies and diabodies) as described, for example,in Milstein et al., NATURE 305:537-539 (1983), WO 93/08829, Trauneckeret al., EMBO J., 10:3655-3659 (1991), WO 94/04690, Suresh et al.,METHODS IN ENZYMOLOGY, 121:210 (1986), WO96/27011, Brennan et al.,SCIENCE, 229: 81 (1985), Shalaby et al., J. EXP. MED., 175: 217-225(1992), Kostelny et al., J. IMMUNOL., 148(5):1547-1553 (1992), Hollingeret al., PNAS, 90:6444-6448, Gruber et al., J. IMMUNOL., 152:5368 (1994),Wu et al., NAT. BIOTECHNOL., 25(11): 1290-1297, U.S. Patent PublicationNo. 2007/0071675, and Bostrom et al., SCIENCE 323:1640-1644 (2009).

In some embodiments, the antibody is conjugated to an effector agentsuch as a small molecule toxin or a radionuclide using standard in vitroconjugation chemistries. If the effector agent is a polypeptide, theantibody can be chemically conjugated to the effector or joined to theeffector as a fusion protein. Construction of fusion proteins is withinordinary skill in the art.

IV. Use of the Antibodies

The antibodies disclosed herein can be used to treat various forms ofcancer, e.g., breast, ovarian, prostate, cervical, colorectal, lung(e.g., non-small cell lung cancer), pancreatic, gastric, skin, kidney,head and neck, and schwannoma cancers. The cancer cells are exposed to atherapeutically effective amount of the antibody so as to inhibit orreduce proliferation of the cancer cell. In some embodiments, theantibodies inhibit cancer cell proliferation by at least 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100%.

In some embodiments, the antibody inhibits or reduces proliferation of atumor cell by inhibiting binding of human ErbB3 to an ErbB3 ligand,e.g., Neuregulin/Heregulin especially NRGβ1/NRG1-β1/NRGβ1/HRGβ1 andNRGα1/NRG1-α1/NRGα1/HRGα1. The antibody can be used in a method toinhibit tumor growth in a human patient. The method comprisesadministering to the patient a therapeutically effective amount of theantibody.

Cancers associated with ErbB3 overexpression and/or activation includebreast cancer, ovarian cancer, prostate cancer, cervical cancer, lungcancer (e.g., non-small cell lung cancer), some forms of brain cancer(e.g., schwannoma), melanomas, skin, kidney, and gastrointestinalcancers (e.g., colorectal, pancreatic, gastric, head and neck).

As used herein, “treat, “treating” and “treatment” mean the treatment ofa disease in a mammal, e.g., in a human. This includes: (a) inhibitingthe disease, i.e., arresting its development; and (b) relieving thedisease, i.e., causing regression of the disease state; and (c) curingthe disease.

Generally, a therapeutically effective amount of active component is inthe range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kgto 10 mg/kg. The amount administered will depend on variables such asthe type and extent of disease or indication to be treated, the overallhealth of the patient, the in vivo potency of the antibody, thepharmaceutical formulation, and the route of administration. The initialdosage can be increased beyond the upper level in order to rapidlyachieve the desired blood-level or tissue level. Alternatively, theinitial dosage can be smaller than the optimum, and the daily dosage maybe progressively increased during the course of treatment. Human dosagecan be optimized, e.g., in a conventional Phase I dose escalation studydesigned to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary,depending on factors such as route of administration, dosage amount andthe disease being treated. Exemplary dosing frequencies are once perday, once per week and once every two weeks. A preferred route ofadministration is parenteral, e.g., intravenous infusion. Formulation ofmonoclonal antibody-based drugs is within ordinary skill in the art. Insome embodiments, the monoclonal antibody is lyophilized andreconstituted in buffered saline at the time of administration.

For therapeutic use, an antibody preferably is combined with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means buffers, carriers, and excipients suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. Thecarrier(s) should be “acceptable” in the sense of being compatible withthe other ingredients of the formulations and not deleterious to therecipient. Pharmaceutically acceptable carriers include buffers,solvents, dispersion media, coatings, isotonic and absorption delayingagents, and the like, that are compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art.

Pharmaceutical compositions containing antibodies disclosed herein canbe presented in a dosage unit form and can be prepared by any suitablemethod. A pharmaceutical composition should be formulated to becompatible with its intended route of administration. Examples of routesof administration are intravenous (IV), intradermal, inhalation,transdermal, topical, transmucosal, and rectal administration. Apreferred route of administration for monoclonal antibodies is IVinfusion. Useful formulations can be prepared by methods well known inthe pharmaceutical art. For example, see Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990). Formulationcomponents suitable for parenteral administration include a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as EDTA; buffers such as acetates, citrates orphosphates; and agents for the adjustment of tonicity such as sodiumchloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol), and suitablemixtures thereof.

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

EXAMPLES

The following Examples are merely illustrative and are not intended tolimit the scope or content of the invention in any way.

Example 1—Production of Anti-hErbB3 Monoclonal Antibodies

Immunizations, fusions, and primary screens were conducted at MaineBiotechnology Services Inc. following the Repetitive ImmunizationMultiple Sites (RIMMS) protocol. Three AJ mice and three Balb/c micewere immunized with recombinant human ErbB3/Fc (R&D Systems, Cat. No.348-RB). Two sets of immunization were performed with either cleavedrhErbB3 (Immunization A) or with cleaved rhErbB3 cross-linked to itsligand, recombinant human NRG1-β1/HRG1-β1-EGF domain (R&D Systems, Cat.No. 396-HB) (Immunization B). Two AJ mice per immunization with seradisplaying high anti-ErbB3 activity by Enzyme Linked Immunosorbent Assay(ELISA) were chosen for subsequent fusion. Spleens and lymph nodes fromthe appropriate mice were harvested. B-cells then were harvested andfused with a myeloma line. Fusion products were serially diluted ontoforty 96-well plates to near clonality. A total of 5280 supernatantsfrom the resulting fusions were screened for binding to recombinantrhErbB3/Fc, using ELISA. The same supernatants were also screened fortheir binding to human ErbB3 overexpressed in CHO cells (by Mesoscaleelectrochemiluminescence assay). Three hundred supernatants identifiedas containing antibodies against ErbB3 were further characterized by invitro biochemical and cell-based assays as discussed below. A panel ofhybridomas was selected, and the hybridomas were subcloned and expanded.Hybridoma cell lines were transferred to BioXCell (formerly Bio-Express)for antibody expression and purification by affinity chromatography onProtein G resin under standard conditions.

Anti-hErbB3 monoclonal antibody 04D01 was generated from Immunization Adescribed above. Anti-hErbB3 monoclonal antibodies 09D03, 11G01, 12A07,18H02, 22A02 and 24C05 were generated from Immunization B describedabove.

Example 2—Sequence Analysis of Anti-hErbB3 Monoclonal Antibodies

The light-chain isotype and heavy chain isotype of each monoclonalantibody in Example 1 was determined using the IsoStrip™ MouseMonoclonal Antibody Isotyping Kit according the manufacturer'sinstructions (Roche Applied Science). All antibodies were determined tobe Kappa light chain and IgG1 or IgG2b IgG heavy chain.

The heavy and light chain variable regions of the mouse monoclonalantibodies were sequenced using 5′ RACE (Rapid Amplification of cDNAEnds). Total RNA was extracted from each monoclonal hybridoma cell lineusing the RNEASY® Miniprep kit according to the vendor's instructions(Qiagen). Full-length first strand cDNA containing 5′ ends was generatedusing either the GENERACER™ Kit (Invitrogen) or SMARTER™ RACE cDNAAmplification Kit (Clontech) according to the manufacturer'sinstructions using random primers for 5′ RACE.

The variable regions of the Kappa and Heavy (IgG1 or IgG2b) IgG chainswere amplified by PCR, using KOD Hot Start Polymerase (Novagen) orAdvantage 2 Polymerase Mix (Clontech) according to the manufacturer'sinstructions. For amplification of 5′ cDNA ends in conjunction with theGENERACER™ Kit, the GENERACER™ 5′ Primer, 5′ cgactggagcacgaggacactga 3′(SEQ ID NO: 136) (Invitrogen) was used as a 5′ primer. For amplificationof 5′ cDNA ends in conjunction with the SMARTER™ RACE™ cDNAAmplification Kit, the Universal Primer Mix A primer (Clontech), a mixof 5′CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3′ (SEQ ID NO: 137)and 5′ CTAATACGACTCACTATAGGGC 3′ (SEQ ID NO: 138), was used as a 5′primer. Heavy chain variable regions were amplified using the above 5′primers and a 3′ IgG1 Constant Region specific primer, either 5′TATGCAAGGCTTACAACCACA 3′ (SEQ ID NO: 139) or 5′GCCAGTGGATAGACAGATGGGGGTGTCG 3′ (SEQ ID NO: 140). IgG2b sequences wereamplified with either 5′ AGGACAGGGGTTGATTGTTGA 3′ (SEQ ID NO: 141), 5′GGCCAGTGGATAGACTGATGGGGGTGTTGT 3′ (SEQ ID NO: 142), or 5′GGAGGAACCAGTTGTATCTCCACACCCA 3′ (SEQ ID NO: 143). Kappa chain variableregions were amplified with the above 5′ primers and a 3′ Kappa ConstantRegion specific primer, either 5′ CTCATTCCTGTTGAAGCTCTTGACAAT 3′ (SEQ IDNO: 144) or 5′ CGACTGAGGCACCTCCAGATGTT 3′ (SEQ ID NO: 145).

Individual PCR products were isolated by agarose gel electrophoresis andpurified using the Qiaquick® Gel Purification kit according to themanufacturer's instructions (Qiagen). The PCR products were subsequentlycloned into the pCR®4Blunt plasmid using the Zero Blunt® TOPO® PCRCloning Kit according to the manufacturer's instructions (Invitrogen)and transformed into DH5-α bacteria (Invitrogen) through standardmolecular biology techniques. Plasmid DNA isolated from transformedbacterial clones was sequenced using M13 Forward (5′ GTAAAACGACGGCCAGT3′) (SEQ ID NO: 146) and M13 Reverse primers (5′ CAGGAAACAGCTATGACC 3′)(SEQ ID NO: 147) by Beckman Genomics, using standard dideoxy DNAsequencing methods to identify the sequence of the variable regionsequences. The sequences were analyzed using Vector NTI software(Invitrogen) and the IMGT/V-Quest software to identify and confirmvariable region sequences.

The nucleic acid sequences encoding and the protein sequences definingvariable regions of the murine monoclonal antibodies are summarizedbelow (amino terminal signal peptide sequences are not shown). CDRsequences (Kabat definition) are shown in bold/underlined in the aminoacid sequences.

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the04D01 Antibody (SEQ ID NO: 1)

1 caggtccaac tgcagcagcc tggggctgaa ctggtgaggc ctgggacttc agtgaagttg 61tcctgcaagg cttctggcta caccttcacc agccactggt tgcactgggt gaagcagagg 121cctggacaag gccttgagtg gatcggagtg cttgatcctt ctgattttta tagtaactac 181aatcaaaact tcaagggcaa ggccacattg actgtagaca catcctccag cacagcctac 241atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc acgaggccta 301ctatccgggg actatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca

Protein Sequence Defining the Heavy Chain Variable Region of the 04D01Antibody (SEQ ID NO: 2)

1 qvqlqqpgae lvrpgtsvkl sckasgytft  shwlh wvkqr pgqglewig v   ldpsdfysny61 nqnfkg katl tvdtssstay mqlssltsed savyycar gl   lsgdyamdyw gqgtsvtvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the04D01 Antibody (SEQ ID NO: 3)

1 gatgttttga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 121tacctgcaga aaccaggcca gtctccaaag tccctgatct acaaagtttc taaccgattt 181tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 241agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc atatgttccg 301tggacgttcg gtggaggcac caagctggaa atcaaa

Protein Sequence Defining the Kappa Chain Variable Region of the 04D01Antibody (SEQ ID NO: 4)

1 dvlmtqipls lpvslgdqas isc rssqsiv   hsngntyle w ylqkpgqspk sliy kvsnrf61 s gvpdrfsgs gsgtdftlki srveaedlgv yyc fqgsyvp   wt fgggtkle ik

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the09D03 Antibody (SEQ ID NO: 11)

1 caggttactc taaaagagtc tggccctggg atattgcggc cctcccagac cctcagtctg 61acttgttctt tctctgggtt ttcactgagc acttttggtt tgagtgtagg ctggattcgt 121cagccttcag ggaagggtct ggagtggctg gcacacattt ggtgggatga tgataagtac 181tataacccag cccttaagag tcggctcaca atctccaagg atacctccaa aaaccaggta 241ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg tgctcgaata 301ggggcggacg cccttccttt tgactactgg ggccaaggca ccactctcac agtctcctca

Protein Sequence Defining the Heavy Chain Variable Region of the 09D03Antibody (SEQ ID NO: 12)

1 qvtlkesgpg ilrpsqtlsl tcsfsgfsls  tfglsvg wir qpsgkglewl a hiwwdddky61 ynpalks rlt iskdtsknqv flkianvdta dtatyycar i   gadalpfdyw gqgttltvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the09D03 Antibody (SEQ ID NO: 13)

1 gatattgtgt tgactcagac tgcaccctct gtacctgtca ctcctggaga gtcagtatcc 61atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 121ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 181tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc 241agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcct 301ttcacgttcg gctcggggac aaagttggaa ataaaa

Protein Sequence Defining the Kappa Chain Variable Region of the 09D03Antibody (SEQ ID NO: 14)

1 divltqtaps vpvtpgesvs isc rssksll   hsngntyly w flqrpgqspq lliy rmsnla61 s gvpdrfsgs gsgtaftlri srveaedvgv yyc mqhleyp   ft fgsgtkle ik

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the11G01 Antibody (SEQ ID NO: 21)

1 caggttcagc tgcaacagtc tgacgctgag ttggtgaaac ctggagcttc agtgaagata 61tcctgcaagg tttctggcta caccttcact gaccatatta ttcactggat gaagcagagg 121cctgaacagg gcctggaatg gattggatat atttatccta gagatggtta tattaagtac 181aatgagaagt tcaagggcaa ggccacattg actgcagaca aatcctccag cacagcctac 241atgcaggtca acagcctgac atctgaggac tctgcagtct atttctgtgc aaggggttac 301tattatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a

Protein Sequence Defining the Heavy Chain Variable Region of the 11G01Antibody (SEQ ID NO: 22)

1 qvqlqqsdae lvkpgasvki sckvsgytft  dhiih wmkqr peqglewig y   iyprdgyiky61 nekfkg katl tadkssstay mqvnsltsed savyfcar gy   yyamdy wgqg tsvtvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the11G01 Antibody (SEQ ID NO: 23)

1 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61atctcttgca gatctagtca gagcattgta catagtattg gaaacaccta tttagaatgg 121tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 181tctggggtcc cagagaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 241agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcca 301ttcacgttcg gctcggggac aaagttggaa ataaaa

Protein Sequence Defining the Kappa Chain Variable Region of the 11G01Antibody (SEQ ID NO: 24)

1 dvlmtqtpls lpvslgdqas isc rssqsiv   hsigntyle w ylqkpgqspk lliy kvsnrf61 sgvperfsgs gsgtdftlki srveaedlgv yyc fqgshvp   ft fgsgtkle ik

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the12A07 Antibody (SEQ ID NO: 30)

1 caggtccaac tgctgcagcc tggggctgag ctggtgaggc ctgggacttc agtgaagttg 61tcctgcaaga cttctggcta caccttctcc agctactgga tgcactgggt aaagcagagg 121cctggacaag gccttgagtg gatcggaatg attgatcctt ctgatgttta tactaactac 181aatccaaagt tcaagggcaa ggccacattg actgttgaca catcctccag cacagcctac 241atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaaactac 301tctggggact actggggcca aggcaccact ctcacagtct cctca

Protein Sequence Defining the Heavy Chain Variable Region of the 12A07Antibody (SEQ ID NO: 31)

1 qvqllqpgae lvrpgtsvkl scktsgytfs  sywmh wvkqr pgqglewig m   idpsdvytny61 npkfkg katl tvdtssstay mqlssltsed savyycar ny   sgdy wgqgtt ltvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the12A07 Antibody (SEQ ID NO: 32)

1 gatgttttga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61atctcttgta gatctagtca gagcattgtc catagtaatg gaaacaccta tttagaatgg 121tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 181tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 241agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc atatgttccg 301tggacgttcg gtggaggcac caagctggaa atcaaa

Protein Sequence Defining the Kappa Chain Variable Region of the 12A07Antibody (SEQ ID NO: 33)

1 dvlmtqipls lpvslgdqas isc rssqsiv   hsngntyle w ylqkpgqspk lliy kvsnrf61 s gvpdrfsgs gsgtdftlki srveaedlgv yyc fqgsyvp   wt fgggtkle ik

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the18H02 Antibody (SEQ ID NO: 37)

1 cagatccagt tggtacagtc tggacctgaa ctgaagaagc ctggagaggc agtcaagatc 61tcctgcaagt cttctgggta taccttcaca acctatggaa tgagctgggt gaaacaggct 121ccaggaaggg ctttaaagtg gatgggctgg ataaacacct actctggagt gccaacatat 181gctgatgact tcaagggacg gtttgccttc tctttggaat cctctgccag cactgcctat 241ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagagggagg 301gatggttacc aagtggcctg gtttgcttac tggggccaag ggacgctggt cactgtctct 361gca

Protein Sequence Defining the Heavy Chain Variable Region of the 18H02Antibody (SEQ ID NO: 38)

1 qiqlvqsgpe lkkpgeavki sckssgytft  tygms wvkqa pgralkwmg w   intysgvpty61 addfkg rfaf slessastay lqinnlkned tatyfcar gr   dgyqvawfay wgqgtlvtvs 121 a

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the18H02 Antibody (SEQ ID NO: 39)

1 gaaacaactg tgacccagtc tccagcatcc ctgtccatgg ctataggaga taaagtcacc 61atcagatgca taaccagcac tgatattgat gatgatatga actggttcca gcagaagcca 121ggggaacctc ctaagctcct tatttcagaa ggcaatactc ttcgtcctgg agtcccatcc 181cgattctccg gcagtggcta tggtacagat tttattttta caattgaaaa catgctctct 241gaagatgttg cagattacta ctgtttgcaa agtgataact tgccgtacac gttcggaggg 301gggaccaagc tggaaataaa a

Protein Sequence Defining the Kappa Chain Variable Region of the 18H02Antibody (SEQ ID NO: 40)

1 ettvtqspas lsmaigdkvt irc itstdid ddmn wfqqkp geppkllis e gntlrpgvps61 rfsgsgygtd fiftienmls edvadyyc lq sdnlpyt fgg gtkleik

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the22A02 Antibody (SEQ ID NO: 47)

1 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctgggacttc agtgaagttg 61tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt aaagcagagg 121cctggacaag gccttgagtg gatcggaatg attgatcctt ctgatagtta tactaactac 181aatccaaagt tcaagggtaa ggccacattg actgtagaca catcctccag cacagcctac 241atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaaactac 301tctggggact actggggcca aggcaccact ctcacagtct cctca

Protein Sequence Defining the Heavy Chain Variable Region of the 22A02Antibody (SEQ ID NO: 48)

1 qvqlqqpgae lvrpgtsvkl sckasgytft  nywmh wvkqr pgqglewig m   idpsdsytny61 npkfkg katl tvdtssstay mqlssltsed savyycar ny   sgdy wgqgtt ltvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the22A02 Antibody (SEQ ID NO: 49)

1 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 121tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 181tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 241agcagagtgg aggctgagga tctgggagtt tattattgct ttcaaggttc atatgttccg 301tggacgttcg gtggaggcac caagctggaa atcaaa

Protein Sequence Defining the Kappa Chain Variable Region of the 22A02Antibody (SEQ ID NO: 50)

1 dvlmtqtpls lpvslgdqas isc rssqsiv   hsngntyle w ylqkpgqspk lliy kvsnrf61 s gvpdrfsgs gsgtdftlki srveaedlgv yyc fqgsyvp   wt fgggtkle ik

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the24C05 Antibody (SEQ ID NO: 53)

1 gaggtgcagc tggtggaatc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 61tcctgtgcag cctctggatt cactttcagt gactatgcca tgtcttgggt tcgccagact 121ccggaaaaga ggctggagtg ggtcgcaacc attagtgatg gtggtactta cacctactat 181ccagacaatg taaagggccg attcaccatc tccagagaca atgccaagaa caacctgtac 241ctgcaaatga gccatctgaa gtctgaggac acagccatgt attactgtgc aagagaatgg 301ggtgattacg acggatttga ctactggggc caaggcacca ctctcacagt ctcctcg

Protein Sequence Defining the Heavy Chain Variable Region of the 24C05Antibody (SEQ ID NO: 54)

1 evqlvesggg lvkpggslkl scaasgftfs  dyams wvrqt pekrlewva t   isdggtytyy61 pdnvkg rfti srdnaknnly lqmshlksed tamyycar ew gdydgfdy wg qgttltvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the24C05 Antibody (SEQ ID NO: 55)

1 gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 61ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca 121gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 181aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcggcag ccttgagtct 241gaagatcttg cagactatta ctgtctacaa tatgatagtt atccgtacac gttcggaggg 301gggaccaagc tggaaataaa a

Protein Sequence Defining the Kappa Chain Variable Region of the 24C05Antibody (SEQ ID NO: 56)

1 diqmtqspss lsaslgervs ltc rasqeis   gyls wlqqkp dgtikrliy a   astldsgvpk 61 rfsgsrsgsd ysltigsles edladyyc lg   ydsypyt fgg gtkleik

The amino acid sequences defining the immunoglobulin heavy chainvariable regions for the antibodies produced in Example 1 are aligned inFIG. 2. Amino terminal signal peptide sequences (for properexpression/secretion) are not shown. CDR₁, CDR₂, and CDR₃ (Kabatdefinition) are identified by boxes. FIG. 3 shows an alignment of theseparate CDR₁, CDR₂, and CDR₃ sequences for each antibody.

The amino acid sequences defining the immunoglobulin light chainvariable regions for the antibodies in Example 1 are aligned in FIG. 4.Amino terminal signal peptide sequences (for properexpression/secretion) are not shown. CDR₁, CDR₂ and CDR₃ are identifiedby boxes. FIG. 5 shows an alignment of the separate CDR₁, CDR₂, and CDR₃sequences for each antibody.

Table 1 is a concordance chart showing the SEQ ID NO. of each sequencediscussed in this Example.

TABLE 1 SEQ. ID NO. Nucleic Acid or Protein 1 04D01 Heavy Chain VariableRegion-nucleic acid 2 04D01 Heavy Chain Variable Region-protein 3 04D01Light (kappa) Chain Variable Region-nucleic acid 4 04D01 Light (kappa)Chain Variable Region-protein 5 04D01 Heavy Chain CDR₁ 6 04D01 HeavyChain CDR₂ 7 04D01 Heavy Chain CDR₃ 8 04D01 Light (kappa) Chain CDR₁ 904D01 Light (kappa) Chain CDR₂ 10 04D01 Light (kappa) Chain CDR₃ 1109D03 Heavy Chain Variable Region-nucleic acid 12 09D03 Heavy ChainVariable Region-protein 13 09D03 Light (kappa) Chain VariableRegion-nucleic acid 14 09D03 Light (kappa) Chain Variable Region-protein15 09D03 Heavy Chain CDR₁ 16 09D03 Heavy Chain CDR₂ 17 09D03 Heavy ChainCDR₃ 18 09D03 Light (kappa) Chain CDR₁ 19 09D03 Light (kappa) Chain CDR₂20 09D03 Light (kappa) Chain CDR₃ 21 11G01 Heavy Chain VariableRegion-nucleic acid 22 11G01 Heavy Chain Variable Region-protein 2311G01 Light (kappa) Chain Variable Region-nucleic acid 24 11G01 Light(kappa) Chain Variable Region-protein 25 11G01 Heavy Chain CDR₁ 26 11G01Heavy Chain CDR₂ 27 11G01 Heavy Chain CDR₃ 28 11G01 Light (kappa) ChainCDR₁ 9 11G01 Light (kappa) Chain CDR₂ 29 11G01 Light (kappa) Chain CDR₃30 12A07 Heavy Chain Variable Region-nucleic acid 31 12A07 Heavy ChainVariable Region-protein 32 12A07 Light (kappa) Chain VariableRegion-nucleic acid 33 12A07 Light (kappa) Chain Variable Region-protein34 12A07 Heavy Chain CDR₁ 35 12A07 Heavy Chain CDR₂ 36 12A07 Heavy ChainCDR₃ 8 12A07 Light (kappa) Chain CDR₁ 9 12A07 Light (kappa) Chain CDR₂10 12A07 Light (kappa) Chain CDR₃ 37 18H02 Heavy Chain VariableRegion-nucleic acid 38 18H02 Heavy Chain Variable Region-protein 3918H02 Light (kappa) Chain Variable Region-nucleic acid 40 18H02 Light(kappa) Chain Variable Region-protein 41 18H02 Heavy Chain CDR₁ 42 18H02Heavy Chain CDR₂ 43 18H02 Heavy Chain CDR₃ 44 18H02 Light (kappa) ChainCDR₁ 45 18H02 Light (kappa) Chain CDR₂ 46 18H02 Light (kappa) Chain CDR₃47 22A02 Heavy Chain Variable Region-nucleic acid 48 22A02 Heavy ChainVariable Region-protein 49 22A02 Light (kappa) Chain VariableRegion-nucleic acid 50 22A02 Light (kappa) Chain Variable Region-protein51 22A02 Heavy Chain CDR₁ 52 22A02 Heavy Chain CDR₂ 36 22A02 Heavy ChainCDR₃ 8 22A02 Light (kappa) Chain CDR₁ 9 22A02 Light (kappa) Chain CDR₂10 22A02 Light (kappa) Chain CDR₃ 53 24C05 Heavy Chain VariableRegion-nucleic acid 54 24C05 Heavy Chain Variable Region-protein 5524C05 Light (kappa) Chain Variable Region-nucleic acid 56 24C05 Light(kappa) Chain Variable Region-protein 57 24C05 Heavy Chain CDR₁ 58 24C05Heavy Chain CDR₂ 59 24C05 Heavy Chain CDR₃ 60 24C05 Light (kappa) ChainCDR₁ 61 24C05 Light (kappa) Chain CDR₂ 62 24C05 Light (kappa) Chain CDR₃

Mouse monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, andIMGT definitions) are shown in Table 2.

TABLE 2 CDR1 CDR2 CDR3 Kabat 04D01 SHWLH VLDPSDFYSNYNQNFKG GLLSGDYAMDY(SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) 09D03 TFGLSVGHIWWDDDKYYNPALKS IGADALPFDY (SEQ ID NO: 15) (SEQ ID NO: 16)(SEQ ID NO: 17) 11G01 DHIIH YIYPRDGYIKYNEKFKG GYYYAMDY (SEQ ID NO: 25)(SEQ ID NO: 26) (SEQ ID NO: 27) 12A07 SYWMH MIDPSDVYTNYNPKFKG NYSGDY(SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) 18H02 TYGMSWINTYSGVPTYADDFKG GRDGYQVAWFAY (SEQ ID NO: 41) (SEQ ID NO: 42)(SEQ ID NO: 43) 22A02 NYWMH MIDPSDSYTNYNPKFKG NYSGDY (SEQ ID NO: 51)(SEQ ID NO: 52) (SEQ ID NO: 36) 24C05 DYAMS TISDGGTYTYYPDNVKG EWGDYDGFDY(SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) Chothia 04D01 GYTFTSHDPSDFY GLLSGDYAMDY (SEQ ID NO: 63) (SEQ ID NO: 64) (SEQ ID NO: 7) 09D03GFSLSTFGL WWDDD IGADALPFDY (SEQ ID NO: 65) (SEQ ID NO: 66)(SEQ ID NO: 17) 11G01 GYTFTDH YPRDGY GYYYAMDY (SEQ ID NO: 67)(SEQ ID NO: 68) (SEQ ID NO: 27) 12A07 GYTFSSY DPSDVY NYSGDY(SEQ ID NO: 69) (SEQ ID NO: 70) (SEQ ID NO: 36) 18H02 GYTFTTY NTYSGVGRDGYQVAWFAY (SEQ ID NO: 71) (SEQ ID NO: 72) (SEQ ID NO: 43) 22A02GYTFTNY DPSDSY NYSGDY (SEQ ID NO: 73) (SEQ ID NO: 74) (SEQ ID NO: 36)24C05 GFTFSDY SDGGTY EWGDYDGFDY (SEQ ID NO: 75) (SEQ ID NO: 76)(SEQ ID NO: 59) IMGT 04D01 GYTFTSHW LDPSDFYS ARGLLSGDYAMDY(SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) 09D03 GFSLSTFGLS IWWDDDKARIGADALPFDY (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82) 11G01GYTFTDHI IYPRDGYI ARGYYYAMDY (SEQ ID NO: 83) (SEQ ID NO: 84)(SEQ ID NO: 85) 12A07 GYTFSSYW IDPSDVYT ARNYSGDY (SEQ ID NO: 86)(SEQ ID NO: 87) (SEQ ID NO: 88) 18H02 GYTFTTYG INTYSGVP ARGRDGYQVAWFAY(SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 91) 22A02 GYTFTNYW IDPSDSYTARNYSGDY (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 88) 24C05 GFTFSDYAISDGGTYT AREWGDYDGFDY (SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 96)

Mouse monoclonal antibody Kappa light chain CDR sequences (Kabat,Chothia, and IMGT definitions) are shown in Table 3.

TABLE 3 CDR1 CDR2 CDR3 Kabat/Chothia 04D01 RSSQSIVHSNGNTYLE KVSNRFSFQGSYVPWT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) 09D03RSSKSLLHSNGNTYLY RMSNLAS MQHLEYPFT (SEQ ID NO: 18) (SEQ ID NO: 19)(SEQ ID NO: 20) 11G01 RSSQSIVHSIGNTYLE KVSNRFS FQGSHVPFT (SEQ ID NO: 28)(SEQ ID NO: 9) (SEQ ID NO: 29) 12A07 RSSQSIVHSNGNTYLE KVSNRFS FQGSYVPWT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) 18H02 ITSTDIDDDMN EGNTLRPLQSDNLPYT (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46) 22A02RSSQSIVHSNGNTYLE KVSNRFS FQGSYVPWT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 10) 24C05 RASQEISGYLS AASTLDS LQYDSYPYT (SEQ ID NO: 60)(SEQ ID NO: 61) (SEQ ID NO: 62) IMGT 04D01 QSIVHSNGNTY KVS FQGSYVPWT(SEQ ID NO: 97) (SEQ ID NO: 10) 09D03 KSLLHSNGNTY RMS MQHLEYPFT(SEQ ID NO: 98) (SEQ ID NO: 20) 11G01 QSIVHSIGNTY KVS FQGSHVPFT(SEQ ID NO: 99) (SEQ ID NO: 29) 12A07 QSIVHSNGNTY KVS FQGSYVPWT(SEQ ID NO: 97) (SEQ ID NO: 10) 18H02 TDIDDD EGN LQSDNLPYT(SEQ ID NO: 100) (SEQ ID NO: 46) 22A02 QSIVHSNGNTY KVS FQGSYVPWT(SEQ ID NO: 97) (SEQ ID NO: 10) 24C05 QEISGY AAS LQYDSYPYT(SEQ ID NO: 101) (SEQ ID NO: 62)

In Tables 2 and 3, the longest CDR sequences for the immunoglobulinheavy chain and light chain are shown in bold.

To create the complete heavy or kappa chain antibody sequences, eachvariable sequence above is combined with its respective constant region.For example, a complete heavy chain comprises a heavy variable sequencefollowed by the murine IgG1 or IgG2b heavy chain constant sequence and acomplete kappa chain comprises a kappa variable sequence followed by themurine kappa light chain constant sequence.

Nucleic Acid Sequence Encoding the Murine IgG Heavy Chain ConstantRegion (SEQ ID NO: 102)

1 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 61tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 121tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 181ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag ccagaccgtc 241acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 301gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 361cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 421gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 481gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 541agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 601aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 661aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 721agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 781aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 841tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 901acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 961tctcctggta aa

Protein Sequence Defining the Murine IgG Heavy Chain Constant Region(SEQ ID NO: 103)

1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd 61lytlsssvtv psstwpsqtv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif 121ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv 181selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv 241sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf 301tcsvlheglh nhhtekslsh spgk

Nucleic Acid Sequence Encoding the Murine IgG2b Heavy Chain ConstantRegion (SEQ ID NO: 104)

1 gccaaaacaa cacccccatc agtctatcca ctggcccctg ggtgtggaga tacaactggt 61tcctctgtga ctctgggatg cctggtcaag ggctacttcc ctgagtcagt gactgtgact 121tggaactctg gatccctgtc cagcagtgtg cacaccttcc cagctctcct gcagtctgga 181ctctacacta tgagcagctc agtgactgtc ccctccagca cctggccaag tcagaccgtc 241acctgcagcg ttgctcaccc agccagcagc accacggtgg acaaaaaact tgagcccagc 301gggcccattt caacaatcaa cccctgtcct ccatgcaagg agtgtcacaa atgcccagct 361cctaacctcg agggtggacc atccgtcttc atcttccctc caaatatcaa ggatgtactc 421atgatctccc tgacacccaa ggtcacgtgt gtggtggtgg atgtgagcga ggatgaccca 481gacgtccaga tcagctggtt tgtgaacaac gtggaagtac acacagctca gacacaaacc 541catagagagg attacaacag tactatccgg gtggtcagca ccctccccat ccagcaccag 601gactggatga gtggcaagga gttcaaatgc aaggtcaaca acaaagacct cccatcaccc 661atcgagagaa ccatctcaaa aattaaaggg ctagtcagag ctccacaagt atacatcttg 721ccgccaccag cagagcagtt gtccaggaaa gatgtcagtc tcacttgcct ggtcgtgggc 781ttcaaccctg gagacatcag tgtggagtgg accagcaatg ggcatacaga ggagaactac 841aaggacaccg caccagtcct agactctgac ggttcttact tcatatatag caagctcaat 901atgaaaacaa gcaagtggga gaaaacagat tccttctcat gcaacgtgag acacgagggt 961ctgaaaaatt actacctgaa gaagaccatc tcccggtctc cgggtaaa

Protein Sequence Defining the Murine IgG2b Heavy Chain Constant Region(SEQ ID NO: 105)

1 akttppsvyp lapgcgdttg ssvtlgclvk gyfpesvtvt wnsgslsssv htfpallqsg 61lytmsssvtv psstwpsqtv tcsvahpass ttvdkkleps gpistinpcp pckechkcpa 121pnleggpsvf ifppnikdvl misltpkvtc vvvdvseddp dvqiswfvnn vevhtaqtqt 181hredynstir vvstlpiqhq dwmsgkefkc kvnnkdlpsp iertiskikg lvrapqvyil 241pppaeqlsrk dvsltclvvg fnpgdisvew tsnghteeny kdtapvldsd gsyfiyskln 301mktskwektd sfscnvrheg lknyylkkti srspgk

Nucleic Acid Sequence Encoding the Murine Kappa Light Chain ConstantRegion (SEQ ID NO: 106)

1 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 61ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccagagacat caatgtcaag 121tggaagattg atggcagtga acgacaaaat ggtgtcctga acagttggac tgatcaggac 181agcaaagaca gcacctacag catgagcagc accctcacat tgaccaagga cgagtatgaa 241cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 301agcttcaaca ggaatgagtg t

Protein Sequence Defining the Murine Kappa Light Chain Constant Region(SEQ ID NO: 107)

1 radaaptvsi fppsseqlts ggasvvcfln nfyprdinvk wkidgserqn gvlnswtdqd 61skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec

The following sequences represent the actual or contemplated full lengthheavy and light chain sequences (i.e., containing both the variable andconstant regions sequences) for each antibody described in this Example.Signal sequences for proper secretion of the antibodies are alsoincluded at the 5′ end of the DNA sequences or the amino terminal end ofthe protein sequences. The variable region sequences can be ligated toother constant region sequences, to produce active full length IgG heavyand light chains.

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG1 Constant Region) of 04D01 (SEQ IDNO: 108)

1 atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacaggtgt ccactcccag 61gtccaactgc agcagcctgg ggctgaactg gtgaggcctg ggacttcagt gaagttgtcc 121tgcaaggctt ctggctacac cttcaccagc cactggttgc actgggtgaa gcagaggcct 181ggacaaggcc ttgagtggat cggagtgctt gatccttctg atttttatag taactacaat 241caaaacttca agggcaaggc cacattgact gtagacacat cctccagcac agcctacatg 301cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcacg aggcctacta 361tccggggact atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctcagcc 421aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 481atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg 541aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc 601tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcca gaccgtcacc 661tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat 721tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc 781ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 841gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg 901cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt 961gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac 1021agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag 1081gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt 1141ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat 1201gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac 1261ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc 1321tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct 1381cctggtaaa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 04D01 (SEQ ID NO:109)

1 mgwsciivll vstatgvhsq vqlqqpgael vrpgtsvkls ckasgytfts hwlhwvkqrp 61gqglewigvl dpsdfysnyn qnfkgkatlt vdtssstaym qlssltseds avyycargll 121sgdyamdywg qgtsvtvssa kttppsvypl apgsaaqtns mvtlgclvkg yfpepvtvtw 181nsgslssgvh tfpavlqsdl ytlsssvtvp sstwpsqtvt cnvahpasst kvdkkivprd 241cgckpcictv pevssvfifp pkpkdvltit ltpkvtcvvv diskddpevq fswfvddvev 301htaqtqpree qfnstfrsvs elpimhqdwl ngkefkcrvn saafpapiek tisktkgrpk 361apqvytippp keqmakdkvs ltcmitdffp editvewqwn gqpaenyknt qpimdtdgsy 421fvysklnvqk snweagntft csvlheglhn hhtekslshs pgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 04D01 (SEQ ID NO:110)

1 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 61gttttgatga cccaaattcc actctccctg cctgtcagtc ttggagatca agcctccatc 121tcttgcagat ctagtcagag cattgtacat agtaatggaa acacctattt agaatggtac 181ctgcagaaac caggccagtc tccaaagtcc ctgatctaca aagtttctaa ccgattttct 241ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 301agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcata tgttccgtgg 361acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 421atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 481aacaacttct accccagaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 541aatggtgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 601agcaccctca cattgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 661actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 04D01 (SEQ ID NO: 111)

1 mklpvrllvl mfwipasssd vlmtqiplsl pvslgdqasi scrssqsivh sngntylewy 61lqkpgqspks liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw 121tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq 181ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG2b Constant Region) of 09D03 (SEQ IDNO: 112)

1 atgggcaggc ttacttcttc attcctgtta ctgattgtcc ctgcatatgt cctgtcccag 61gttactctaa aagagtctgg ccctgggata ttgcggccct cccagaccct cagtctgact 121tgttctttct ctgggttttc actgagcact tttggtttga gtgtaggctg gattcgtcag 181ccttcaggga agggtctgga gtggctggca cacatttggt gggatgatga taagtactat 241aacccagccc ttaagagtcg gctcacaatc tccaaggata cctccaaaaa ccaggtattc 301ctcaagatcg ccaatgtgga cactgcagat actgccacat actactgtgc tcgaataggg 361gcggacgccc ttccttttga ctactggggc caaggcacca ctctcacagt ctcctcagcc 421aaaacaacac ccccatcagt ctatccactg gcccctgggt gtggagatac aactggttcc 481tccgtgacct ctgggtgcct ggtcaagggg tacttccctg agccagtgac tgtgacttgg 541aactctggat ccctgtccag cagtgtgcac accttcccag ctctcctgca gtctggactc 601tacactatga gcagctcagt gactgtcccc tccagcacct ggccaagtca gaccgtcacc 661tgcagcgttg ctcacccagc cagcagcacc acggtggaca aaaaacttga gcccagcggg 721cccatttcaa caatcaaccc ctgtcctcca tgcaaggagt gtcacaaatg cccagctcct 781aacctcgagg gtggaccatc cgtcttcatc ttccctccaa atatcaagga tgtactcatg 841atctccctga cacccaaggt cacgtgtgtg gtggtggatg tgagcgagga tgacccagac 901gtccagatca gctggtttgt gaacaacgtg gaagtacaca cagctcagac acaaacccat 961agagaggatt acaacagtac tatccgggtg gtcagcaccc tccccatcca gcaccaggac 1021tggatgagtg gcaaggagtt caaatgcaag gtgaacaaca aagacctccc atcacccatc 1081gagagaacca tctcaaaaat taaagggcta gtcagagctc cacaagtata cactttgccg 1141ccaccagcag agcagttgtc caggaaagat gtcagtctca cttgcctggt cgtgggcttc 1201aaccctggag acatcagtgt ggagtggacc agcaatgggc atacagagga gaactacaag 1261gacaccgcac cagttcttga ctctgacggt tcttacttca tatatagcaa gctcaatatg 1321aaaacaagca agtgggagaa aacagattcc ttctcatgca acgtgagaca cgagggtctg 1381aaaaattact acctgaagaa gaccatctcc cggtctccgg gtaaa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG2b Constant Region) of 09D03 (SEQ ID NO:113)

1 mgrltssfll livpayvlsq vtlkesgpgi lrpsqtlslt csfsgfslst fglsvgwirq 61psgkglewla hiwwdddkyy npalksrlti skdtsknqvf lkianvdtad tatyycarig 121adalpfdywg qgttltvssa kttppsvypl apgcgdttgs svtsgclvkg yfpepvtvtw 181nsgslsssvh tfpallqsgl ytmsssvtvp sstwpsqtvt csvahpasst tvdkklepsg 241pistinpcpp ckechkcpap nleggpsvfi fppnikdvlm isltpkvtcv vvdvseddpd 301vqiswfvnnv evhtaqtqth redynstirv vstlpiqhqd wmsgkefkck vnnkdlpspi 361ertiskikgl vrapqvytlp ppaeqlsrkd vsltclvvgf npgdisvewt snghteenyk 421dtapvldsdg syfiysklnm ktskwektds fscnvrhegl knyylkktis rspgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 09D03 (SEQ ID NO:114)

1 atgaggtgcc tagctgagtt cctggggctg cttgtgctct ggatccctgg agccattggg 61gatattgtgt tgactcagac tgcaccctct gtacctgtca ctcctggaga gtcagtatcc 121atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 181ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 241tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc 301agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcct 361ttcacgttcg gctcggggac aaagttggaa ataaaacggg ctgatgctgc accaactgta 421tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 481ttgaacaact tctaccccag agacatcaat gtcaagtgga agattgatgg cagtgaacga 541caaaatggtg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 601agcagcaccc tcacattgac caaggacgag tatgaacgac ataacagcta tacctgtgag 661gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgagtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 09D03 (SEQ ID NO: 115)

1 mrclaeflgl lvlwipgaig divltqtaps vpvtpgesvs iscrssksll hsngntylyw 61flqrpgqspq lliyrmsnla sgvpdrfsgs gsgtaftlri srveaedvgv yycmqhleyp 121ftfgsgtkle ikradaaptv sifppsseql tsggasvvcf lnnfyprdin vkwkidgser 181qngvlnswtd qdskdstysm sstltltkde yerhnsytce athktstspi vksfnrnec

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG1 Constant Region) of 11G01 (SEQ IDNO: 116)

1 atggaatgga gctgggtctc tctcttcttc ctgtcagtaa ctacaggtgt ccactcccag 61gttcagctgc aacagtctga cgctgagttg gtgaaacctg gagcttcagt gaagatatcc 121tgcaaggttt ctggctacac cttcactgac catattattc actggatgaa gcagaggcct 181gaacagggcc tggaatggat tggatatatt tatcctagag atggttatat taagtacaat 241gagaagttca agggcaaggc cacattgact gcagacaaat cctccagcac agcctacatg 301caggtcaaca gcctgacatc tgaggactct gcagtctatt tctgtgcaag gggttactat 361tatgctatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagc caaaacgaca 421cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 481ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 541tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 601agcagctcag tgactgtccc ctccagcacc tggcccagcc agaccgtcac ctgcaacgtt 661gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt 721aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc 781aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc 841aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 901cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc 961atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct 1021ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag 1081gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc 1141atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca 1201gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac 1261agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg 1321ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 11G01 (SEQ ID NO:117)

1 mewswvslff lsvttgvhsq vqlqqsdael vkpgasvkis ckvsgytftd hiihwmkqrp 61eqglewigyi yprdgyikyn ekfkgkatlt adkssstaym qvnsltseds avyfcargyy 121yamdywgqgt svtvssaktt ppsvyplapg saaqtnsmvt lgclvkgyfp epvtvtwnsg 181slssgvhtfp avlqsdlytl sssvtvpsst wpsqtvtcnv ahpasstkvd kkivprdcgc 241kpcictvpev ssvfifppkp kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta 301qtqpreeqfn stfrsvselp imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq 361vytipppkeq makdkvsltc mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy 421sklnvqksnw eagntftcsv lheglhnhht ekslshspgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 11G01 (SEQ ID NO:118)

1 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagaagtgat 61gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 121tcttgcagat ctagtcagag cattgtacat agtattggaa acacctattt agaatggtac 181ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 241ggggtcccag agaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 301agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc 361acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgcacc aactgtatcc 421atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 481aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 541aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 601agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 661actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 11G01 (SEQ ID NO: 119)

1 mklpvrllvl mfwipasrsd vlmtqtplsl pvslgdqasi scrssqsivh signtylewy 61lqkpgqspkl liykvsnrfs gvperfsgsg sgtdftlkis rveaedlgvy ycfqgshvpf 121tfgsgtklei kradaaptvs ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq 181ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG Constant Region) of 12A07 (SEQ IDNO: 120)

1 atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacatgtgt ccactcccag 61gtccaactgc tgcagcctgg ggctgagctg gtgaggcctg ggacttcagt gaagttgtcc 121tgcaagactt ctggctacac cttctccagc tactggatgc actgggtaaa gcagaggcct 181ggacaaggcc ttgagtggat cggaatgatt gatccttctg atgtttatac taactacaat 241ccaaagttca agggcaaggc cacattgact gttgacacat cctccagcac agcctacatg 301cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactactct 361ggggactact ggggccaagg caccactctc acagtctcct cagccaaaac gacaccccca 421tctgtctatc cactggcccc tggatctgct gcccaaacta actccatggt gaccctggga 481tgcctggtca agggctattt ccctgagcca gtgacagtga cctggaactc tggatccctg 541tccagcggtg tgcacacctt cccagctgtc ctgcagtctg acctctacac tctgagcagc 601tcagtgactg tcccctccag cacctggccc agccagaccg tcacctgcaa cgttgcccac 661ccggccagca gcaccaaggt ggacaagaaa attgtgccca gggattgtgg ttgtaagcct 721tgcatatgta cagtcccaga agtatcatct gtcttcatct tccccccaaa gcccaaggat 781gtgctcacca ttactctgac tcctaaggtc acgtgtgttg tggtagacat cagcaaggat 841gatcccgagg tccagttcag ctggtttgta gatgatgtgg aggtgcacac agctcagacg 901caaccccggg aggagcagtt caacagcact ttccgctcag tcagtgaact tcccatcatg 961caccaggact ggctcaatgg caaggagttc aaatgcaggg tcaacagtgc agctttccct 1021gcccccatcg agaaaaccat ctccaaaacc aaaggcagac cgaaggctcc acaggtgtac 1081accattccac ctcccaagga gcagatggcc aaggataaag tcagtctgac ctgcatgata 1141acagacttct tccctgaaga cattactgtg gagtggcagt ggaatgggca gccagcggag 1201aactacaaga acactcagcc catcatggac acagatggct cttacttcgt ctacagcaag 1261ctcaatgtgc agaagagcaa ctgggaggca ggaaatactt tcacctgctc tgtgttacat 1321gagggcctgc acaaccacca tactgagaag agcctctccc actctcctgg taaa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 12A07 (SEQ ID NO:121)

1 mgwsciivll vstatcvhsq vqllqpgael vrpgtsvkls cktsgytfss ywmhwvkqrp 61gqglewigmi dpsdvytnyn pkfkgkatlt vdtssstaym qlssltseds avyycarnys 121gdywgqgttl tvssakttpp svyplapgsa aqtnsmvtlg clvkgyfpep vtvtwnsgsl 181ssgvhtfpav lqsdlytlss svtvpsstwp sqtvtcnvah passtkvdkk ivprdcgckp 241cictvpevss vfifppkpkd vltitltpkv tcvvvdiskd dpevqfswfv ddvevhtaqt 301qpreeqfnst frsvselpim hqdwlngkef kcrvnsaafp apiektiskt kgrpkapqvy 361tipppkeqma kdkvsltcmi tdffpeditv ewqwngqpae nykntqpimd tdgsyfvysk 421lnvqksnwea gntftcsvlh eglhnhhtek slshspgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 12A07 (SEQ ID NO:122)

1 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 61gttttgatga cccaaattcc actctccctg cctgtcagtc ttggagatca agcctccatc 121tcttgtagat ctagtcagag cattgtccat agtaatggaa acacctattt agaatggtac 181ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 241ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 301agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcata tgttccgtgg 361acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 421atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 481aacaacttct accccagaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 541aatggtgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 601agcaccctca cattgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 661actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 12A07 (SEQ ID NO: 123)

1 mklpvrllvl mfwipasssd vlmtqiplsl pvslgdqasi scrssqsivh sngntylewy 61lqkpgqspkl liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw 121tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq 181ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG Constant Region) of 18H02 (SEQ IDNO: 124)

1 atgggttggc tgtggaactt gctattcctg atggcagctg cccaaagtgc ccaagcacag 61atccagttgg tacagtctgg acctgaactg aagaagcctg gagaggcagt caagatctcc 121tgcaagtctt ctgggtatac cttcacaacc tatggaatga gctgggtgaa acaggctcca 181ggaagggctt taaagtggat gggctggata aacacctact ctggagtgcc aacatatgct 241gatgacttca agggacggtt tgccttctct ttggaatcct ctgccagcac tgcctatttg 301cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag agggagggat 361ggttaccaag tggcctggtt tgcttactgg ggccaaggga cgctggtcac tgtctctgca 421gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 481tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 541tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 601ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag ccagaccgtc 661acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 721gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 781cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 841gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 901gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 961agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 1021aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 1081aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 1141agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 1201aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 1261tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 1321acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 1381tctcctggta aatga

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 18H02 (SEQ ID NO:125)

1 mgwlwnllfl maaaqsaqaq iqlvqsgpel kkpgeavkis ckssgytftt ygmswvkqap 61gralkwmgwi ntysgvptya ddfkgrfafs lessastayl qinnlknedt atyfcargrd 121gyqvawfayw gqgtlvtvsa akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt 181wnsgslssgv htfpavlqsd lytlsssvtv psstwpsqtv tcnvahpass tkvdkkivpr 241dcgckpcict vpevssvfif ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve 301vhtaqtqpre eqfnstfrsv selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp 361kapqvytipp pkeqmakdkv sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs 421yfvysklnvq ksnweagntf tcsvlheglh nhhtekslsh spgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 18H02 (SEQ ID NO:126)

1 atgttctcac tagctcttct cctcagtctt cttctcctct gtgtctctga ttctagggca 61gaaacaactg tgacccagtc tccagcatcc ctgtccatgg ctataggaga taaagtcacc 121atcagatgca taaccagcac tgatattgat gatgatatga actggttcca gcagaagcca 181ggggaacctc ctaagctcct tatttcagaa ggcaatactc ttcgtcctgg agtcccatcc 241cgattctccg gcagtggcta tggtacagat tttattttta caattgaaaa catgctctct 301gaagatgttg cagattacta ctgtttgcaa agtgataact tgccgtacac gttcggaggg 361gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 421tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 481cccagagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggtgtcctg 541aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcaca 601ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 661tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 18H02 (SEQ ID NO: 127)

1 mfslalllsl lllcvsdsra ettvtqspas lsmaigdkvt ircitstdid ddmnwfqqkp 61geppkllise gntlrpgvps rfsgsgygtd fiftienmls edvadyyclq sdnlpytfgg 121gtkleikrad aaptvsifpp sseqltsgga svvcflnnfy prdinvkwki dgserqngvl 181nswtdqdskd stysmsstlt ltkdeyerhn sytceathkt stspivksfn rnec

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG Constant Region) of 22A02 (SEQ IDNO: 128)

1 atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacaggtgt ccactcccag 61gtccaactgc agcagcctgg ggctgagctg gtgaggcctg ggacttcagt gaagttgtcc 121tgcaaggctt ctggctacac cttcaccaac tactggatgc actgggtaaa gcagaggcct 181ggacaaggcc ttgagtggat cggaatgatt gatccttctg atagttatac taactacaat 241ccaaagttca agggtaaggc cacattgact gtagacacat cctccagcac agcctacatg 301cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactactct 361ggggactact ggggccaagg caccactctc acagtctcct cagccaaaac gacaccccca 421tctgtctatc cactggcccc tggatctgct gcccaaacta actccatggt gaccctggga 481tgcctggtca agggctattt ccctgagcca gtgacagtga cctggaactc tggatccctg 541tccagcggtg tgcacacctt cccagctgtc ctgcagtctg acctctacac tctgagcagc 601tcagtgactg tcccctccag cacctggccc agccagaccg tcacctgcaa cgttgcccac 661ccggccagca gcaccaaggt ggacaagaaa attgtgccca gggattgtgg ttgtaagcct 721tgcatatgta cagtcccaga agtatcatct gtcttcatct tccccccaaa gcccaaggat 781gtgctcacca ttactctgac tcctaaggtc acgtgtgttg tggtagacat cagcaaggat 841gatcccgagg tccagttcag ctggtttgta gatgatgtgg aggtgcacac agctcagacg 901caaccccggg aggagcagtt caacagcact ttccgctcag tcagtgaact tcccatcatg 961caccaggact ggctcaatgg caaggagttc aaatgcaggg tcaacagtgc agctttccct 1021gcccccatcg agaaaaccat ctccaaaacc aaaggcagac cgaaggctcc acaggtgtac 1081accattccac ctcccaagga gcagatggcc aaggataaag tcagtctgac ctgcatgata 1141acagacttct tccctgaaga cattactgtg gagtggcagt ggaatgggca gccagcggag 1201aactacaaga acactcagcc catcatggac acagatggct cttacttcgt ctacagcaag 1261ctcaatgtgc agaagagcaa ctgggaggca ggaaatactt tcacctgctc tgtgttacat 1321gagggcctgc acaaccacca tactgagaag agcctctccc actctcctgg taaa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 22A02 (SEQ ID NO:129)

1 mgwsciivll vstatgvhsq vqlqqpgael vrpgtsvkls ckasgytftn ywmhwvkqrp 61gqglewigmi dpsdsytnyn pkfkgkatlt vdtssstaym qlssltseds avyycarnys 121gdywgqgttl tvssakttpp svyplapgsa aqtnsmvtlg clvkgyfpep vtvtwnsgsl 181ssgvhtfpav lqsdlytlss svtvpsstwp sqtvtcnvah passtkvdkk ivprdcgckp 241cictvpevss vfifppkpkd vltitltpkv tcvvvdiskd dpevqfswfv ddvevhtaqt 301qpreeqfnst frsvselpim hqdwlngkef kcrvnsaafp apiektiskt kgrpkapqvy 361tipppkeqma kdkvsltcmi tdffpeditv ewqwngqpae nykntqpimd tdgsyfvysk 421lnvqksnwea gntftcsvlh eglhnhhtek slshspgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 22A02 (SEQ ID NO:130)

1 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 61gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 121tcttgcagat ctagtcagag cattgtacat agtaatggaa acacctattt agaatggtac 181ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 241ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 301agagtggagg ctgaggatct gggagtttat tattgctttc aaggttcata tgttccgtgg 361acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 421atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 481aacaacttct accccagaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 541aatggtgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 601agcaccctca cattgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 661actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain

1 mklpvrllvl mfwipasssd vlmtqtplsl pvslgdqasi scrssqsivh sngntylewy 61lqkpgqspkl liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw 121tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq 181ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG Constant Region) of 24C05 (SEQ IDNO: 132)

1 atgaacttcg ggctcagctt gatgttcctt gtccttgtct taaaaggtgt ccagtgtgag 61gtgcagctgg tggaatctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc 121tgtgcagcct ctggattcac tttcagtgac tatgccatgt cttgggttcg ccagactccg 181gaaaagaggc tggagtgggt cgcaaccatt agtgatggtg gtacttacac ctactatcca 241gacaatgtaa agggccgatt caccatctcc agagacaatg ccaagaacaa cctgtacctg 301caaatgagcc atctgaagtc tgaggacaca gccatgtatt actgtgcaag agaatggggt 361gattacgacg gatttgacta ctggggccaa ggcaccactc tcacagtctc ctcggccaaa 421acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg 481gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac 541tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac 601actctgagca gctcagtgac tgtcccctcc agcacctggc ccagccagac cgtcacctgc 661aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt 721ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca 781aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac 841atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac 901acagctcaga cgcaaccccg ggaggagcag ttcaacagca ctttccgctc agtcagtgaa 961cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt 1021gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct 1081ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg 1141acctgcatga taacagactt cttccctgaa gacattactg tggagtggca gtggaatggg 1201cagccagcgg agaactacaa gaacactcag cccatcatgg acacagatgg ctcttacttc 1261gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc 1321tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct 1381ggtaaa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 24C05 (SEQ ID NO:133)

1 mnfglslmfl vlvlkgvqce vqlvesgggl vkpggslkls caasgftfsd yamswvrqtp 61ekrlewvati sdggtytyyp dnvkgrftis rdnaknnlyl qmshlksedt amyycarewg 121dydgfdywgq gttltvssak ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn 181sgslssgvht fpavlqsdly tlsssvtvps stwpsqtvtc nvahpasstk vdkkivprdc 241gckpcictvp evssvfifpp kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh 301taqtqpreeq fnstfrsvse lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka 361pqvytipppk eqmakdkvsl tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf 421vysklnvqks nweagntftc svlheglhnh htekslshsp gk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 24C05 (SEQ ID NO:134)

1 atggacatga gggttcctgc tcacgttttt ggcttcttgt tgctctggtt tccaggtacc 61agatgtgaca tccagatgac ccagtctcca tcctccttat ctgcctctct gggagaaaga 121gtcagtctca cttgtcgggc aagtcaggaa attagtggtt acttaagctg gcttcagcag 181aaaccagatg gaactattaa acgcctgatc tacgccgcat ccactttaga ttctggtgtc 241ccaaaaaggt tcagtggcag taggtctggg tcagattatt ctctcaccat cggcagcctt 301gagtctgaag atcttgcaga ctattactgt ctacaatatg atagttatcc gtacacgttc 361ggagggggga ccaagctgga aataaaacgg gctgatgctg caccaactgt atccatcttc 421ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac 481ttctacccca gagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggt 541gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc 601ctcacattga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac 661aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 24C05 (SEQ ID NO: 135)

1 mdmrvpahvf gflllwfpgt rcdiqmtqsp sslsaslger vsltcrasqe isgylswlqq 61kpdgtikrli yaastldsgv pkrfsgsrsg sdysltigsl esedladyyc lqydsypytf 121gggtkleikr adaaptvsif ppsseqltsg gasvvcflnn fyprdinvkw kidgserqng 181vlnswtdqds kdstysmsst ltltkdeyer hnsytceath ktstspivks fnrnec

For convenience, Table 4 provides a concordance chart showing thecorrespondence between the full length sequences of the antibodiesdiscussed in this Example with those presented in the Sequence Listing.

TABLE 4 SEQ ID NO. Nucleic Acid or Protein 108 04D01 Heavy Variable +IgG1 Constant-nucleic acid 109 04D01 Heavy Variable + IgG1Constant-protein 110 04D01 Kappa Variable + Constant-nucleic acid 11104D01 Kappa Variable + Constant-protein 112 09D03 Heavy Variable + IgG2bConstant-nucleic acid 113 09D03 Heavy Variable + IgG2b Constant-protein114 09D03 Kappa Variable + Constant-nucleic acid 115 09D03 KappaVariable + Constant-protein 116 11G01 Heavy Variable + IgG1Constant-nucleic acid 117 11G01 Heavy Variable + IgG1 Constant-protein118 11G01 Kappa Variable + Constant-nucleic acid 119 11G01 KappaVariable + Constant-protein 120 12A07 Heavy Variable + IgG1Constant-nucleic acid 121 12A07 Heavy Variable + IgG1 Constant-protein122 12A07 Kappa Variable + Constant-nucleic acid 123 12A07 KappaVariable + Constant-protein 124 18H02 Heavy Variable + IgG1Constant-nucleic acid 125 18H02 Heavy Variable + IgG1 Constant-protein126 18H02 Kappa Variable + Constant-nucleic acid 127 18H02 KappaVariable + Constant-protein 128 22A02 Heavy Variable + IgG1Constant-nucleic acid 129 22A02 Heavy Variable + IgG1 Constant-protein130 22A02 Kappa Variable + Constant-nucleic acid 131 22A02 KappaVariable + Constant-protein 132 24C05 Heavy Variable + IgG1Constant-nucleic acid 133 24C05 Heavy Variable + IgG1 Constant-protein134 24C05 Kappa Variable + Constant-nucleic acid 135 24C05 KappaVariable + Constant-protein

Example 3—Binding Affinities

The binding affinities and kinetics of the binding of monoclonalantibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 torecombinant human ErbB3/Fc fusion protein (rhErbB3-Fc) were measured bysurface plasmon resonance using a BIACORE® T100 (a BIACORE®) instrument.

Rabbit anti-mouse IgGs (a BIACORE®, Cat. No. BR-1008-38) wereimmobilized on carboxymethylated dextran CM4 sensor chips (a BIACORE®,Cat. No. BR-1005-34) by amine coupling (a BIACORE®, Cat. No. BR-1000-50)using a standard coupling protocol according to vendor's instructions.The analyses were performed at 25° C., using PBS (Invitrogen, Cat. No.14040-133) containing 0.05% surfactant P20 (a BIACORE®, Cat. No.BR-1000-54) as running buffer.

The antibodies were captured in individual flow cells at a flow rate of10 μl/minute. Injection time was varied for each antibody to yield anR_(max) between 30 and 60 RU. Buffer or rhErbB3-Fc diluted in runningbuffer was injected sequentially over a reference surface (no antibodycaptured) and the active surface (antibody to be tested) for 300 secondsat 60 μl/minute. The dissociation phase was monitored for up to 3600seconds. The surface was then regenerated with two 60-seconds injectionof 10 mM Glycine-HCl, pH 1.7 (made from Glycine pH 1.5 (a BIACORE®, Cat.No. BR-1003-54) and pH 2.0 (a BIACORE®, Cat. No. BR-1003-55)) at a flowrate of 60 μl/minute. The rhErbB3-Fc concentration range tested was0.125 nM to 20 nM.

Kinetic parameters were determined using the kinetic function of theBIAevaluation software (Biacore) with double reference subtraction.Kinetic parameters for each antibody, k_(a) (association rate constant),k_(d) (dissociation rate constant) and K_(D) (equilibrium dissociationconstant) were determined. Kinetic values of the monoclonal antibodieson rhErbB3-Fc at 25C are summarized in Table 5.

TABLE 5 Standard Standard Standard Antibody k_(a) (1/Ms) Deviation k_(d)(1/s) Deviation K_(D) (M) Deviation n 04D01 3.8E+05 3.0E+04 9.3E−051.9E−05 2.5E−10 5.6E−11 5 09D03 2.7E+05 3.2E+04 2.0E−05 1.2E−05 8.0E−115.5E−11 3 11G01 2.7E+05 9.2E+04 2.2E−05 9.6E−06 9.1E−11 5.5E−11 4 12A076.2E+05 8.1E+04 1.9E−04 1.0E−04 3.0E−10 1.4E−10 3 18H02 2.8E+05 3.1E+042.5E−05 8.8E−06 9.1E−11 3.7E−11 4 22A02 7.0E+05 8.1E+04 2.2E−04 1.4E−043.2E−10 2.4E−10 3 24C05 1.5E+06 2.0E+05 9.2E−06 3.0E−06 6.5E−12 2.8E−124

The data in Table 5 demonstrate that the antibodies bind rhErbB3 with aK_(D) of about 350 pM or less, 250 pM or less, 200 pM or less, 150 pM orless, 100 pM or less, 50 pM or less, or 10 pM or less.

Example 4—Neutralization Activity

In this example, the antibodies produced in Example 1 were tested forability to inhibit rhErbB3 binding to NRG1-β1 and NRG1-α1. Theantibodies were tested by electrochemiluminescence (ECL) assay forinhibition of hErbB3 binding to NRG1-β1. MA2400 96-well standard bindingplates (Meso Scale Discovery, Cat. No. L15XA-6) were coated with 50 μlof 0.5 μg/mL rhErbB3/Fc (R&D systems, Cat. No. 348-RB) in PBS(Invitrogen, Cat. No. 14040-133) for overnight at 4° C. with noagitation. The plates then were washed 3 times with PBS+0.1% Tween20(Sigma P5927) and blocked with 200 μl of PBS containing 5% BSA (SeraCare Life Sciences, Cat. No. AP-4510-80) for 1.5 hour at roomtemperature. After washing the plates 3 times with PBS, 25 μl of theantibody dilutions were added to the plates for another hour at roomtemperature with agitation. Ligand NRG1-β1 (R&D Systems, Cat. No.377-HB, 26 kDa) was added to the wells at the final concentration of0.25 μg/ml. The plates were washed three times with PBS and incubatedwith 25 μl of 1 μg/mL biotinylated antibody against human NRG1-β1 (R&Dsystems, Cat. No BAF377) preincubated for one hour with SULTO-TAGStreptavidin (Meso Scale Discovery, Cat. No R32AD-5) for one hour atroom temperature with agitation. The plates then were washed 3 timeswith PBS, and 150 μl of 1× read buffer (Meso Scale Discovery, Cat. No.R92TC-1) was added to each well before the plates were analyzed on aSector® Imager 2400 (Meso Scale Discovery) instrument.

The interaction of NRG1-β1 with ErbB3 was inhibited by antibodies 04D01,12A07, 18H02, 22A02 and 24C05 (FIG. 6A). The interaction of NRG1-β1 withrhErbB3 was enhanced by antibody 09D03, but not as well as by antibody11G01 (FIG. 6B).

The murine anti-human ErbB3 antibody IC₅₀ values for neutralization ofNRG1-β1 binding to rhErbB3 for the antibodies (i.e., 04D01, 12A07,18H02, 22A02 and 24C05) were calculated and are summarized in Table 6.

TABLE 6 IC₅₀ (nM) Standard Antibody Average Deviation n 04D01 0.22320.0711 4 12A07 0.2351 0.0530 4 18H02 0.3460 0.0873 4 22A02 0.2418 0.07554 24C05 0.3367 0.0764 4

The results show that antibodies 04D01, 12A07, 18H02, 22A02, and 24C05efficiently neutralized NRG1-β1 binding to rhErbB3. Antibodies 09D03 and11G01 enhanced hNRG1-β1 binding to hErbB3.

The antibodies were tested by ECL assay for inhibition of hErbB3 bindingto the second ErbB3 ligand, NRG1-α1. To assay inhibition of binding ofNRG1-α1 to rhErbB3, the same method used for NRG1-β1 was used, exceptfor the following changes: concentrations of plated rhErbB3/Fc (R&D4518-RB) and of ligand NRG1-al (Thermo Scientific, RP-317-β1AX) were 1μg/ml and 1.5 μg/ml, respectively.

The interaction of NRG1-al with rhErbB3 was inhibited by 11G01, 12A07,18H02, 22A02, and 24C05 IgG1, and was enhanced by antibody 09D03 (FIG.7).

Example 5—Binding to ErbB3 Domain II

In this example, the antibodies produced in Example 1 were tested forbinding to the dimerization domain (domain 2) of hErbB3-ECD. Domain 2 ofhErbB3 (118 amino acids, position 210-327) was cloned in place of domain2 of Her2 (119 amino acids, position AA220-338) into the full-lengthHer2 receptor. The hybrid construct Her2/3d2 was cloned into pLenti6.3and packaged by transient transfection of 293T cells into a Lentivirususing the ViraPower™ Lentiviral Support Kit (Invitrogen, Cat. No.K497000). CHO cells were infected with the lentivirus expressing theHer2/3d2 hybrid protein. The binding of the anti-ErbB3 hybridomasupernatants to Her2/3d2 were tested on these engineered CHO cells byECL with sulfo-tagged anti-mouse antibodies. Data on the binding of thehybridoma supernatants to the chimeric protein Her2/3d2 expressed on thecell surface of CHO cells are summarized in FIG. 8. These results showthat antibodies 09D03 and 11G01 bound to the ErbB3 domain II, AA210-327.

Example 6—Anti-Proliferative Activity

This example describes a characterization of the antibodies produced inExample 1 for their ability to inhibit NRG1-β1 dependent proliferationof cells. Antibodies were tested in the BaF/3 cell system engineered toexpress both human Her2 and ErbB3 and in the human MCF7 breast cancercells which naturally express both Her2 and ErbB3 and grow in responseto NRG1-β1 stimulation.

BaF/3 cells were infected by two lentiviruses engineered to expresshuman Her2 or human ErbB3. Infected cells were selected with blasticidin(15 μg/ml; Invitrogen, Cat. No. R21001) and individual colonies wereisolated and tested for expression of both receptors. Her2/ErbB3expressing clones were maintained in culture under blasticidin selectionwith [80% RPMI Medium 1640 (GIBCO, Cat. No. 11875-093), 10% fetal bovineserum (GIBCO, Cat. No. 10438-026) and 10% WEHI cell conditioned media{90% ISCOVE's Modified Dulbecco's Medium (GIBCO, Cat. No. 12440053), 10%fetal bovine serum (GIBCO, Cat. No. 10438-026)+2 mM L-glutamine (GIBCO,Cat. No. 25030-081)+0.0025 mM mercaptoethanol (Invitrogen, Cat. No.21985-023)}]. To screen for antagonistic ErbB3 antibodies, cells wererinsed with PBS, and grown in the absence of blasticidin and WEHIconditioned media. Assays were conducted in a 96-well plate (5,000cells/well) in the presence of NRG1-31 (100 ng/ml) and variousconcentrations of antibodies (0.018-5000 ng/ml in 100 μl final volume).MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assays were conducted 3-4 days post NRG1-β1 stimulation.

An example of the dose-dependent inhibition of NRG1-β1 dependent cellproliferation of Her2/ErbB3-BaF/3 by murine anti-human ErbB3 antibodiesis shown in FIG. 9. Inhibition data of NRG1-β1 dependentHer2/ErbB3-BaF/3 cell line proliferation with monoclonal antibodies(i.e., 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05) aresummarized in Table 7.

TABLE 7 Her2/ErbB3-BaF/3, NRG1-β1 dep. Proliferation Antibody IC₅₀(nM)-Average Standard deviation n 04D01 0.373 0.061 3 09D03 1.395 0.2683 11G01 1.934 0.116 3 12A07 0.854 0.059 3 18H02 1.930 0.276 3 22A021.291 0.151 3 24C05 0.145 0.031 3

The results in Table 7 show that antibodies 04D01, 09D03, 11G01, 12A07,18H02, 22A02 and 24C05 strongly inhibited NRG1-β1-induced proliferationof BaF/3 cells expressing Her2/ErbB3.

MCF7 cells (ATCC, Cat. No. HTB-22) were maintained as recommended byATCC. Cells were plated at 5,000 cells/well in a 96-well plate. Cellswere starved overnight in the absence of serum. The following day,NRG1-β1 (40 ng/ml) and various concentrations of antibodies (12.8pg/ml-20 μg/ml in 100 μl final volume) were added to the cells. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assayswere conducted three days post NRG1-β1 stimulation.

An example of the dose-dependent inhibition of NRG1-β1 dependentproliferation of MCF7 cells by murine anti-human ErbB3 antibodies isshown in FIG. 10. Inhibition data of NRG1-β1 dependent MCF7 cellproliferation with antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02and 24C05 are summarized in Table 8.

TABLE 8 MCF7 cells, NRG1-β1 dependent Proliferation Antibody IC₅₀(nM)-Average Standard deviation n 04D01 0.47 0.23 3 09D03 2.28 0.60 311G01 1.98 1.34 3 12A07 0.74 0.48 3 18H02 1.00 0.20 3 22A02 1.62 0.60 324C05 0.39 0.04 3

The results in Table 8 demonstrate that antibodies 04D01, 09D03, 11G01,12A07, 18H02, 22A02, and 24C05 strongly inhibited NRG1-β1-inducedproliferation of MCF7 cells.

The antibodies produced in Example 1 were also tested for their abilityto inhibit proliferation of ErbB3 expressing human cancer cells. Breastcancer cells SKBR-3 overexpress Her2 and are sensitive to Her2-specificinhibitory antibodies.

SKBR-3 cells (ATCC, Cat. No. HTB-30) were maintained as recommended byATCC. Cells were plated at 5,000 cells/well in a 96-well plate in thepresence of 5 μg/ml of antibodies but without exogenous NRG1-β1. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assayswere conducted after three days in culture.

An example of inhibition of cell proliferation of SKBR-3 cells by murineanti-human ErbB3 antibodies is shown in FIG. 11. The results in FIG. 11show that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05inhibited proliferation of SKBR-3 cells.

Example 7—Inhibition of Downstream Signaling

This example describes a characterization of the antibodies produced inExample 1 for their ability to inhibit NRG1-β1 dependent phosphorylationof ErbB3 and the downstream kinase Akt, as the readout for PI3Kactivation. These antibodies were also tested for their ability toinhibit steady state phosphorylation of ErbB3 and Akt in exponentiallygrowing cells.

Breast cancer cells SKBR-3 and MCF7 and prostate cancer cells DU145 weremaintained as recommended by ATCC. Cells were starved overnight in 0%FBS, treated for one hour with 5 μg/ml of antibody followed by NRG1-β1stimulation. Lysates were either analyzed by ELISA with thePhospho-ErbB3 kit from R&D Systems (Cat. No DYC1769) or with thePhospho-Akt ELISA kit from Cell Signaling (Cat. No 7143).

An example of the inhibition of the NRG1-β1 induced phosphorylation ofErbB3 in SKBR-3 cells by murine anti-human ErbB3 antibodies is shown inFIG. 12. The results in FIG. 12 demonstrated that antibodies 04D01,09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited at least 50% ofthe phosphorylation of ErbB3 induced by NRG1-31 in SKBR-3 cells.

An example of the inhibition of the NRG1- 31 induced phosphorylation ofAkt in MCF7 and DU145 cells by murine anti-human ErbB3 antibodies isshown in FIG. 13A and FIG. 13B, respectively. The results in FIGS. 13Aand 13B demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02,22A02 and 24C05 inhibited at least 80% of the phosphorylation of Akt inresponse to the NRG1-31 in both MCF7 and DU145 cells.

The capacity of the anti-ErbB3 antibodies to inhibit the steady statephosphorylation status of ErbB3 and Akt in a breast cancer cell lineSKBR-3 and a pancreatic cancer cell line BxPC3 were tested by treatingthese exponentially growing cells for one hour in presence of antibodiesat 5 μg/ml.

Western blot analysis of these experiments demonstrated that antibodies04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited the steadystate level of phosphorylation of Akt and ErbB3 in both SKBR-3 and BxPC3cells.

Example 8—Inhibition of NRG1-β1-Induced EGFR Phosphorylation

In this example, the antibodies produced in Example 1 were tested fortheir ability to inhibit NRG1-β1 dependent phosphorylation of EGFR inthe ovarian cancer cell line NCI/ADR-RES. NCI/ADR-RES cells (DTP/DCTDNCI tumor repository) were starved overnight in 0% FBS, pre-treated withantibody (5 μg/ml) for one hour followed by NRG1-β1 (20 ng/ml)stimulation for 15 minutes. The phosphorylation of EGFR on tyrosine 1068was analyzed by Western blot. The results of this experimentdemonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02and 24C05 inhibited the phosphorylation of EGFR in response to theNRG1-β1 in NCI/ADR-RES cells.

Example 9—Inhibition of EGF-Induced ErbB3 Phosphorylation

In this example, the antibodies produced in Example 1 were tested fortheir ability to inhibit EGF dependent phosphorylation of ErbB3 in theEGFR overexpressing, epidermoid cancer cell line A431. A431 cells (ATCC,Cat. No CRL-1555) were starved overnight in 0% FBS, pre-treated withantibody (5 μg/ml) for one hour followed by EGF (R&D Systems, Cat. No.236-EG) (50 ng/ml) stimulation for 15 minutes. The phosphorylation ofErbB3 was analyzed by Western blot. The results of this experimentdemonstrated that antibodies 04D01, 09D03, 12A07, 18H02, 22A02 and 24C05inhibited to various extents the phosphorylation of ErbB3 in response tothe EGF in A431 cells.

Example 10—Inhibition of NRG1-β1-Induced her2/ErbB3 HeterodimerFormation

This example describes a characterization of the antibodies produced inExample 1 for their ability to inhibit the formation of the Her2/ErbB3dimer in response to NRG1-β1 in SKBR-3 cells. Breast cancer cells SKBR-3were starved overnight in 0% FBS, treated for one hour with 5 μg/ml ofantibody followed by NRG1-β1 stimulation (30 ng/ml, 30 min). Lysateswere immunoprecipitated with anti-Her2 antibody (R&D Systems, Cat. No.BAF1129) and analyzed by Western blot with polyclonal anti-ErbB3antibody (Santa Cruz, Cat. No. SC285).

The results of this experiment demonstrated that antibodies 04D01,09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited NRG1-β1-inducedHer2/ErbB3 dimer formation in SKBR-3 cells.

Example 11—Inhibition of BxPC3 Tumor Xenograft Growth

The ability of murine monoclonal antibodies produced in Example 1 toinhibit tumor growth was tested in a pancreatic BxPC3 xenograft model.Human pancreatic BxPC3 cells were grown in culture in 37° C. in anatmosphere containing 5% CO2, using RMPI medium containing 10% fetalbovine serum. BxPC3 cells were inoculated subcutaneously into the flankof 8-week old female CB.17 SCID mice (Taconic Labs) with 10×10⁶ cellsper mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumormeasurements were taken twice weekly using vernier calipers. Tumorvolume was calculated using the formula: width×width×length/2. Whentumors reached approximately 200 mm³, the mice were randomized into 9groups of 10 mice each. One group received PBS and another receivedhuman IgG control (huIgG). Each of the other eight groups received oneof the antibody, 04D01, 09D03, 18H02, 11G01, 24C05, 22A02, or 12A07. Allantibodies were dosed at 20 mg/kg body weight, twice per week, byintra-peritoneal injection for 6 weeks. Tumor volumes and mouse bodyweights were recorded twice per week. Tumor growth inhibition wasanalyzed using ANOVA and is expressed as percent inhibition compared tothe PBS control.

The results in FIG. 14 show that antibody 24C05 inhibited tumor growthby 76% in this model (p<0.001). Antibodies 04D01, 18H02 and 11G01 alsoinhibited tumor growth in this model at 64%, 71%, and 72%, respectively(p<0.001). Antibodies 12A07 and 22A02 demonstrated the least activity,i.e., near 40% tumor growth inhibition, while antibody 09D03 gave 60%tumor growth inhibition in this model.

Example 12—Humanization of Anti-ErbB3 Antibodies

A. Construction of Humanized and Chimeric Anti-ErbB3 Antibodies

This Example describes the humanization of the murine antibodydesignated 24C05, and the characterization of the resulting humanizedantibodies. The humanized anti-ErbB3 antibodies were designed using theSUPERHUMANIZATION™ method (Arana Therapeutics Ltd. and Hwang, W. Y. etal. (2005) METHODS 36:35-42) or the CDR grafting method with backmutations (some human framework residues were changed to murineresidues) (See e.g., U.S. Pat. Nos. 5,530,101; 5,693,761; 5,693,762;5,585,089; 6,180,370; 7,022,500). With the exception of heavy chainCDR1, the Kabat CDR definitions were used for CDR grafting onto humanframeworks. A combination of Kabat and Chothia definitions were used forgrafting heavy CDR1. The designed amino acid sequences were converted tocodon-optimized DNA sequences and synthesized by DNA2.0, Inc. to include(in the following order): 5′ HindIII restriction site, Kozak consensussequence, amino terminal signal sequence, humanized variable region,human IgG1 or Kappa constant region, stop codon, and a 3′ EcoRIrestriction site. Additionally, one humanized heavy chain, Sh24C05Hv3-11 Heavy IgG1, was mutated using overlap extension PCR to enhancehumanization, resulting in the Sh24C05 Hv3-11 N62S heavy chain IgG1. Ahuman IgG2 version of the Sh24C05 Hv3-11 N62S heavy chain was alsoconstructed.

The anti-ErbB3 antibody chains humanized according to theSUPERHUMANIZATION™ method, as described herein, are designated with theprefix “Sh” before the antibody chain name. The anti-ErbB3 antibodychains humanized by the CDR grafting method with back mutations, asdescribed herein, are designated with the prefix “Hu” before theantibody chain name.

Chimeric (murine variable region and human constant region) 24C05 heavy(human IgG1) and light (human Kappa) chains were also constructed. Themurine variable regions were fused to the human constant region usingoverlap extension PCR, including (in the following order): 5′ HindIIIrestriction site, Kozak consensus sequence, amino terminal signalsequence, mouse variable region, human IgG1 or Kappa constant region,stop codon, and 3′ EcoRI restriction site.

The humanized and chimeric heavy chains were subcloned into pEE6.4(Lonza Biologics) via HindIII and EcoRI sites using IN-FUSION™ PCRcloning (Clontech). The humanized and chimeric Kappa light chains weresubcloned into pEE14.4 (Lonza Biologics) via HindIII and EcoRI sitesusing IN-FUSION™ PCR cloning.

Humanized antibody chains or chimeric antibody chains were transientlytransfected into 293T cells to produce antibody. Antibody was eitherpurified or used in cell culture media supernatant for subsequent invitro analysis. Binding of the chimeric and humanized antibodies tohuman ErbB3 was measured as described below. The results are summarizedin Table 15.

Additionally, some humanized antibody heavy and light chain combinationswere stably expressed in CHOK1SV cells using the GS SYSTEM™ (LonzaBiologics) in order to produce large quantities of purified humanizedantibody. A single expression vector was constructed by combining pEE6.4and pEE14.4 based vectors. First, pEE6.4 containing full lengthhumanized heavy chain cDNA was digested with NotI and SalI to isolatethe hCMV-MIE promoter+full length humanized heavy chain cDNA+SV40 polyAfragment. This fragment was inserted into the pEE14.4 vector alreadycontaining full length humanized light chain cDNA via NotI/SalI sites,thus creating an expression vector that simultaneously expresses heavyand light chains. The combined heavy and light chain vector waslinearized and transfected into CHOK1SV cells. Stable clones wereselected in the presence of methionine sulfoximine.

Each of the possible combinations of the humanized immunoglobulin heavychain and immunoglobulin light chain variable regions are set forthbelow in Table 9.

TABLE 9 Light Chain Variable Region Heavy Chain Variable Region Hu24C05KvA (SEQ ID NO: 174) Hu24C05 HvA (SEQ ID NO: 162) Hu24C05 KvA (SEQ IDNO: 174) Sh24C05 Hv3-21 (SEQ ID NO: 156) Hu24C05 KvA (SEQ ID NO: 174)Sh24C05 Hv3-23 (SEQ ID NO: 158) Hu24C05 KvA (SEQ ID NO: 174) Sh24C05Hv3-30 (SEQ ID NO: 160) Hu24C05 KvA (SEQ ID NO: 174) Sh24C05 Hv3-7 (SEQID NO: 150) Hu24C05 KvA (SEQ ID NO: 174) Sh24C05 Hv3-11 (SEQ ID NO: 152)Hu24C05 KvA (SEQ ID NO: 174) Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)Sh24C05 Kv1-16 (SEQ ID NO: 166) Hu24C05 HvA (SEQ ID NO: 162) Sh24C05Kv1-16 (SEQ ID NO: 166) Sh24C05 Hv3-21 (SEQ ID NO: 156) Sh24C05 Kv1-16(SEQ ID NO: 166) Sh24C05 Hv3-23 (SEQ ID NO: 158) Sh24C05 Kv1-16 (SEQ IDNO: 166) Sh24C05 Hv3-30 (SEQ ID NO: 160) Sh24C05 Kv1-16 (SEQ ID NO: 166)Sh24C05 Hv3-7 (SEQ ID NO: 150) Sh24C05 Kv1-16 (SEQ ID NO: 166) Sh24C05Hv3-11 (SEQ ID NO: 152) Sh24C05 Kv1-16 (SEQ ID NO: 166) Sh24C05 Hv3-11N62S (SEQ ID NO: 154) Sh24C05 Kv1-17 (SEQ ID NO: 168) Hu24C05 HvA (SEQID NO: 162) Sh24C05 Kv1-17 (SEQ ID NO: 168) Sh24C05 Hv3-21 (SEQ ID NO:156) Sh24C05 Kv1-17 (SEQ ID NO: 168) Sh24C05 Hv3-23 (SEQ ID NO: 158)Sh24C05 Kv1-17 (SEQ ID NO: 168) Sh24C05 Hv3-30 (SEQ ID NO: 160) Sh24C05Kv1-17 (SEQ ID NO: 168) Sh24C05 Hv3-7 (SEQ ID NO: 150) Sh24C05 Kv1-17(SEQ ID NO: 168) Sh24C05 Hv3-11 (SEQ ID NO: 152) Sh24C05 Kv1-17 (SEQ IDNO: 168) Sh24C05 Hv3-11 N62S (SEQ ID NO: 154) Sh24C05 Kv1-33 (SEQ ID NO:170) Hu24C05 HvA(SEQ ID NO: 162) Sh24C05 Kv1-33 (SEQ ID NO: 170) Sh24C05Hv3-21 (SEQ ID NO: 156) Sh24C05 Kv1-33 (SEQ ID NO: 170) Sh24C05 Hv3-23(SEQ ID NO: 158) Sh24C05 Kv1-33 (SEQ ID NO: 170) Sh24C05 Hv3-30 (SEQ IDNO: 160) Sh24C05 Kv1-33 (SEQ ID NO: 170) Sh24C05 Hv3-7 (SEQ ID NO: 150)Sh24C05 Kv1-33 (SEQ ID NO: 170) Sh24C05 Hv3-11 (SEQ ID NO: 152) Sh24C05Kv1-33 (SEQ ID NO: 170) Sh24C05 Hv3-11 N62S (SEQ ID NO: 154) Sh24C05Kv1-9 (SEQ ID NO: 164) Hu24C05 HvA(SEQ ID NO: 162) Sh24C05 Kv1-9 (SEQ IDNO: 164) Sh24C05 Hv3-21 (SEQ ID NO: 156) Sh24C05 Kv1-9 (SEQ ID NO: 164)Sh24C05 Hv3-23 (SEQ ID NO: 158) Sh24C05 Kv1-9 (SEQ ID NO: 164) Sh24C05Hv3-30 (SEQ ID NO: 160) Sh24C05 Kv1-9 (SEQ ID NO: 164) Sh24C05 Hv3-7(SEQ ID NO: 150) Sh24C05 Kv1-9 (SEQ ID NO: 164) Sh24C05 Hv3-11 (SEQ IDNO: 152) Sh24C05 Kv1-9 (SEQ ID NO: 164) Sh24C05 Hv3-11 N62S (SEQ ID NO:154) Sh24C05 Kv1-39 (SEQ ID NO: 172) Hu24C05 HvA (SEQ ID NO: 162)Sh24C05 Kv1-39 (SEQ ID NO: 172) Sh24C05 Hv3-21 (SEQ ID NO: 156) Sh24C05Kv1-39 (SEQ ID NO: 172) Sh24C05 Hv3-23 (SEQ ID NO: 158) Sh24C05 Kv1-39(SEQ ID NO: 172) Sh24C05 Hv3-30 (SEQ ID NO: 160) Sh24C05 Kv1-39 (SEQ IDNO: 172) Sh24C05 Hv3-7 (SEQ ID NO: 150) Sh24C05 Kv1-39 (SEQ ID NO: 172)Sh24C05 Hv3-11 (SEQ ID NO: 152) Sh24C05 Kv1-39 (SEQ ID NO: 172) Sh24C05Hv3-11 N62S (SEQ ID NO: 154)

The nucleic acid sequences encoding and the protein sequences definingvariable regions of the humanized 24C05 antibodies are summarized below(amino terminal signal peptide sequences are not shown). CDR sequences(Kabat definition) are shown in bold and are underlined in the aminoacid sequences.

Nucleic Acid Sequence Encoding the Sh24C05 Hv3-7 Heavy Chain VariableRegion (SEQ ID NO: 149)

1 gaggttcagc tggtggaatc tggcggtggg cttgtacaac caggaggctc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121cccgggaaag gactggagtg ggttgccact atcagcgatg gcggaacgta tacctattac 181cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac 241ctgcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Sh24C05 Hv3-7 Heavy Chain Variable Region(SEQ ID NO: 150)

1 evqlvesggg lvqpggslrl scaasgftfs  dyams wvrqa pgkglewva t isdggtytyy61 pdnvkg rfti srdnaknsly lqmnslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Sh24C05 Hv3-11 Heavy Chain VariableRegion (SEQ ID NO: 151)

1 caagttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatggat caggcaagca 121cccgggaaag gactggagtg ggttagcact atcagcgatg gcggaacgta tacctattac 181cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac 241cttcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Sh24C05 Hv3-11 Heavy Chain Variable Region(SEQ ID NO: 152)

1 qvqlvesggg lvkpggslrl scaasgftfs  dyams wirqa pgkglewvs t isdggtytyy61 pdnvkg rfti srdnaknsly lqmnslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Sh24C05 Hv3-11 N62S Heavy ChainVariable Region (SEQ ID NO: 153)

1 caagttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatggat caggcaagca 121cccgggaaag gactggagtg ggttagcact atcagcgatg gcggaacgta tacctattac 181cctgactccg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac 241cttcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Sh24C05 Hv3-11 N62S Heavy Chain VariableRegion (SEQ ID NO: 154)

1 qvqlvesggg lvkpggslrl scaasgftfs  dyams wirqa pgkglewvs t isdggtytyy61 pdsvkg rfti srdnaknsly lqmnslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Sh24C05 Hv3-21 Heavy Chain VariableRegion (SEQ ID NO: 155)

1 gaggttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121cccgggaaag gactggagtg ggttagcact atcagcgatg gcggaacgta tacctattac 181cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctat 241ttgcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Sh24C05 Hv3-21 Heavy Chain Variable Region(SEQ ID NO: 156)

1 evqlvesggg lvkpggslrl scaasgftfs  dyams wvrqa pgkglewvs t isdggtytyy61 pdnvkg rfti srdnaknsly lqmnslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Sh24C05 Hv3-23 Heavy Chain VariableRegion (SEQ ID NO: 157)

1 gaggttcagc ttctggaatc tggcggtggg cttgtacagc caggaggctc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121cccgggaaag gactggagtg ggtttcaact atcagcgatg gcggaacgta tacctattac 181cctgacaatg tgaagggtcg gttcaccatt tccagggata acagcaagaa cacactctat 241ctccagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Sh24C05 Hv3-23 Heavy Chain Variable Region(SEQ ID NO: 158)

1 evqllesggg lvqpggslrl scaasgftfs  dyams wvrqa pgkglewvs t isdggtytyy61 pdnvkg rfti srdnskntly lqmnslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Sh24C05 Hv3-30 Heavy Chain VariableRegion (SEQ ID NO: 159)

1 caggttcagc tggtggaatc tggcggtggg gtagtacaac caggacggtc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121cccgggaaag gactggagtg ggttgccact atcagcgatg gcggaacgta tacctattac 181cctgacaatg tgaagggtcg gttcaccatt tccagggata actcaaagaa caccctctat 241ctccaaatga gtagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Sh24C05 Hv3-30 Heavy Chain Variable Region(SEQ ID NO: 160)

1 qvqlvesggg vvqpgrslrl scaasgftfs  dyams wvrqa pgkglewva t isdggtytyy61 pdnvkg rfti srdnskntly lqmsslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Hu24C05 HvA Heavy Chain VariableRegion (SEQ ID NO: 161)

1 gaggttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg 61agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121cccgggaaag gactggagtg ggttgccact atcagcgatg gcggaacgta tacctattac 181cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac 241cttcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct

Protein Sequence Defining the Hu24C05 HvA Heavy Chain Variable Region(SEQ ID NO: 162)

1 evqlvesggg lvkpggslrl scaasgftfs  dyams wvrqa pgkglewva t isdggtytyy61 pdnvkg rfti srdnaknsly lqmnslraed tavyycar ew gdydgfdy wg qgtlvtvss

Nucleic Acid Sequence Encoding the Sh24C05 Kv1-9 Kappa Chain VariableRegion (SEQ ID NO: 163)

1 gatattcagt tgacccaatc acctagcttc ctctcagctt ccgtgggcga cagagttacc 61ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtacca acagaagccc 121ggaaaagccc ctaagctgtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181cgattctccg gttctggctc cggaacagag ttcactctga caatttctag ccttcagcca 241gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301ggcactaaac tggagatcaa a

Protein Sequence Defining the Sh24C05 Kv1-9 Kappa Chain Variable Region(SEQ ID NO: 164)

1 diqltqspsf lsasvgdrvt itc rasqeis gyls wyqqkp gkapklliy a astlds gvps61 rfsgsgsgte ftltisslqp edfatyyc lq ydsypyt fgq gtkleik

Nucleic Acid Sequence Encoding the Sh24C05 Kv1-16 Kappa Chain VariableRegion (SEQ ID NO: 165)

1 gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc 61ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtttca acagaagccc 121ggaaaggccc cgaagagctt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181cgattctccg gttctggctc cggaaccgac tttactctga caatttctag ccttcagcca 241gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301ggcactaaac tggagatcaa a

Protein Sequence Defining the Sh24C05 Kv1-16 Kappa Chain Variable Region(SEQ ID NO: 166)

1 diqmtqspss lsasvgdrvt itc rasqeis gyls wfqqkp gkapksliy a astlds gvps61 rfsgsgsgtd ftltisslqp edfatyyc lq ydsypyt fgq gtkleik

Nucleic Acid Sequence Encoding the Sh24C05 Kv1-17 Kappa Chain VariableRegion (SEQ ID NO: 167)

1 gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc 61ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtatca acagaagccc 121ggaaaagccc caaagaggtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181cgattctccg gttctggctc cggaaccgag ttcactctga caatttctag ccttcagcca 241gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301ggcactaaac tggagatcaa a

Protein Sequence Defining the Sh24C05 Kv1-17 Kappa Chain Variable Region(SEQ ID NO: 168)

1 digmtqspss lsasvgdrvt itc rasqeis gyls wyqqkp gkapkrliy a astlds gvps61 rfsgsgsgte ftltisslqp edfatyyc lq ydsypyt fgq gtkleik

Nucleic Acid Sequence Encoding the Sh24C05 Kv1-33 Kappa Chain VariableRegion (SEQ ID NO: 169)

1 gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc 61ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtacca acagaagccc 121ggaaaggccc ccaagctgtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181cgattctccg gttctggctc cggaacagac tttactttta caatttctag ccttcagcca 241gaggacatcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301ggcactaaac tggagatcaa a

Protein Sequence Defining the Sh24C05 Kv1-33 Kappa Chain Variable Region(SEQ ID NO: 170)

1 diqmtqspss lsasvgdrvt itc rasqeis gyls wyqqkp gkapklliy a astlds gvps61 rfsgsgsgtd ftftisslqp ediatyyc lq ydsypyt fgq gtkleik

Nucleic Acid Sequence Encoding the Sh24C05 Kv1-39 Kappa Chain VariableRegion (SEQ ID NO: 171)

1 gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc 61ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtatca acagaagccc 121ggaaaagccc ctaagctgtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181cgattctccg gttctggctc cggaactgac ttcactctga caatttctag ccttcagcca 241gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301ggcactaaac tggagatcaa a

Protein Sequence Defining the Sh24C05 Kv1-39 Kappa Chain Variable Region(SEQ ID NO: 172)

1 diqmtqspss lsasvgdrvt itc rasqeis gyls wyqqkp gkapklliy a astlds gvps61 rfsgsgsgtd ftltisslqp edfatyyc lq ydsypyt fgq gtkleik

Nucleic Acid Sequence Encoding the Hu24C05 KvA Kappa Chain VariableRegion (SEQ ID NO: 173)

1 gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc 61ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggctgca acagaagccc 121ggaggcgcca tcaagaggtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181cgattctccg gttctggctc cggaagtgac tacactctga caatttctag ccttcagcca 241gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301ggcactaaac tggagatcaa a

Protein Sequence Defining the Hu24C05 KvA Kappa Chain Variable Region(SEQ ID NO: 174)

1 diqmtqspss lsasvgdrvt itc rasqeis gyls wlqqkp ggaikrliy a astlds gvps61 rfsgsgsgsd ytltisslqp edfatyyc lq ydsypyt fgq gtkleik

The amino acid sequences defining the immunoglobulin heavy chainvariable regions for the antibodies produced in Example 12 are alignedin FIG. 15. Amino terminal signal peptide sequences (for properexpression/secretion) are not shown. CDR₁, CDR₂, and CDR₃ (Kabatdefinition) are identified by boxes.

The amino acid sequences defining the immunoglobulin light chainvariable regions for the antibodies in Example 12 are aligned in FIG.16. Amino terminal signal peptide sequences (for properexpression/secretion) are not shown. CDR₁, CDR₂ and CDR₃ are identifiedby boxes.

Table 10 is a concordance chart showing the SEQ ID NO. of each sequencediscussed in this Example.

TABLE 10 SEQ. ID NO. Nucleic Acid or Protein 149 Sh24C05 Hv3-7 HeavyChain Variable Region - nucleic acid 150 Sh24C05 Hv3-7 Heavy ChainVariable Region - protein 57 Sh24C05 Hv3-7 Heavy Chain CDR₁ 58 Sh24C05Hv3-7 Heavy Chain CDR₂ 59 Sh24C05 Hv3-7 Heavy Chain CDR₃ 151 Sh24C05Hv3-11 Heavy Chain Variable Region - nucleic acid 152 Sh24C05 Hv3-11Heavy Chain Variable Region - protein 57 Sh24C05 Hv3-11 Heavy Chain CDR₁58 Sh24C05 Hv3-11 Heavy Chain CDR₂ 59 Sh24C05 Hv3-11 Heavy Chain CDR₃153 Sh24C05 Hv3-11 N62S Heavy Chain Variable Region - nucleic acid 154Sh24C05 Hv3-11 N62S Heavy Chain Variable Region - protein 57 Sh24C05Hv3-11 N62S Heavy Chain CDR₁ 148 Sh24C05 Hv3-11 N62S Heavy Chain CDR₂ 59Sh24C05 Hv3-11 N62S Heavy Chain CDR₃ 155 Sh24C05 Hv3-21 Heavy ChainVariable Region - nucleic acid 156 Sh24C05 Hv3-21 Heavy Chain VariableRegion - protein 57 Sh24C05 Hv3-21 Heavy Chain CDR₁ 58 Sh24C05 Hv3-21Heavy Chain CDR₂ 59 Sh24C05 Hv3-21 Heavy Chain CDR₃ 157 Sh24C05 Hv3-23Heavy Chain Variable Region - nucleic acid 158 Sh24C05 Hv3-23 HeavyChain Variable Region - protein 57 Sh24C05 Hv3-23 Heavy Chain CDR₁ 58Sh24C05 Hv3-23 Heavy Chain CDR₂ 59 Sh24C05 Hv3-23 Heavy Chain CDR₃ 159Sh24C05 Hv3-30 Heavy Chain Variable Region - nucleic acid 160 Sh24C05Hv3-30 Heavy Chain Variable Region - protein 57 Sh24C05 Hv3-30 HeavyChain CDR₁ 58 Sh24C05 Hv3-30 Heavy Chain CDR₂ 59 Sh24C05 Hv3-30 HeavyChain CDR₃ 161 Hu24C05 HvA Heavy Chain Variable Region - nucleic acid162 Hu24C05 HvA Heavy Chain Variable Region - protein 57 Hu24C05 HvAHeavy Chain CDR₁ 58 Hu24C05 HvA Heavy Chain CDR₂ 59 Hu24C05 HvA HeavyChain CDR₃ 163 Sh24C05 Kv1-9 Light (kappa) Chain Variable Region -nucleic acid 164 Sh24C05 Kv1-9 Light (kappa) Chain Variable Region -protein 60 Sh24C05 Kv1-9 Light (kappa) Chain CDR₁ 61 Sh24C05 Kv1-9 Light(kappa) Chain CDR₂ 62 Sh24C05 Kv1-9 Light (kappa) Chain CDR₃ 165 Sh24C05Kv1-16 Light (kappa) Chain Variable Region - nucleic acid 166 Sh24C05Kv1-16 Light (kappa) Chain Variable Region - protein 60 Sh24C05 Kv1-16Light (kappa) Chain CDR₁ 61 Sh24C05 Kv1-16 Light (kappa) Chain CDR₂ 62Sh24C05 Kv1-16 Light (kappa) Chain CDR₃ 167 Sh24C05 Kv1-17 Light (kappa)Chain Variable Region - nucleic acid 168 Sh24C05 Kv1-17 Light (kappa)Chain Variable Region - protein 60 Sh24C05 Kv1-17 Light (kappa) ChainCDR₁ 61 Sh24C05 Kv1-17 Light (kappa) Chain CDR₂ 62 Sh24C05 Kv1-17 Light(kappa) Chain CDR₃ 169 Sh24C05 Kv1-33 Light (kappa) Chain VariableRegion - nucleic acid 170 Sh24C05 Kv1-33 Light (kappa) Chain VariableRegion - protein 60 Sh24C05 Kv1-33 Light (kappa) Chain CDR₁ 61 Sh24C05Kv1-33 Light (kappa) Chain CDR₂ 62 Sh24C05 Kv1-33 Light (kappa) ChainCDR₃ 171 Sh24C05 Kv1-39 Light (kappa) Chain Variable Region - nucleicacid 172 Sh24C05 Kv1-39 Light (kappa) Chain Variable Region - protein 60Sh24C05 Kv1-39 Light (kappa) Chain CDR₁ 61 Sh24C05 Kv1-39 Light (kappa)Chain CDR₂ 62 Sh24C05 Kv1-39 Light (kappa) Chain CDR₃ 173 Hu24C05 KvALight (kappa) Chain Variable Region - nucleic acid 174 Hu24C05 KvA Light(kappa) Chain Variable Region - protein 60 Hu24C05 KvA Light (kappa)Chain CDR₁ 61 Hu24C05 KvA Light (kappa) Chain CDR₂ 62 Hu24C05 KvA Light(kappa) Chain CDR₃

Humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia,and IMGT definitions) are shown in Table 11.

TABLE 11 CDR1 CDR2 CDR3 Kabat 24C05 DYAMS TISDGGTYTYYPDNVKG EWGDYDGFDY(SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) Sh24C05 DYAMSTISDGGTYTYYPDNVKG EWGDYDGFDY Hv3-7 (SEQ ID NO: 57) (SEQ ID NO: 58)(SEQ ID NO: 59) Sh24C05 DYAMS TISDGGTYTYYPDNVKG EWGDYDGFDY Hv3-11(SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) Sh24C05 DYAMSTISDGGTYTYYPDSVKG EWGDYDGFDY Hv3-11 (SEQ ID NO: 57) (SEQ ID NO: 148)(SEQ ID NO: 59) N62S Sh24C05 DYAMS TISDGGTYTYYPDNVKG EWGDYDGFDY Hv3-21(SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) Sh24C05 DYAMSTISDGGTYTYYPDNVKG EWGDYDGFDY Hv3-23 (SEQ ID NO: 57) (SEQ ID NO: 58)(SEQ ID NO: 59) Sh24C05 DYAMS TISDGGTYTYYPDNVKG EWGDYDGFDY Hv3-30(SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) Hu24C05 DYAMSTISDGGTYTYYPDNVKG EWGDYDGFDY HvA (SEQ ID NO: 57) (SEQ ID NO: 58)(SEQ ID NO: 59) Chothia 24C05 GFTFSDY SDGGTY EWGDYDGFDY (SEQ ID NO: 75)(SEQ ID NO: 76) (SEQ ID NO: 59) Sh24C05 GFTFSDY SDGGTY EWGDYDGFDY Hv3-7(SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 59) Sh24C05 GFTFSDY SDGGTYEWGDYDGFDY Hv3-11 (SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 59)Sh24C05 GFTFSDY SDGGTY EWGDYDGFDY Hv3-11 (SEQ ID NO: 75) (SEQ ID NO: 76)(SEQ ID NO: 59) N62S Sh24C05 GFTFSDY SDGGTY EWGDYDGFDY Hv3-21(SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 59) Sh24C05 GFTFSDY SDGGTYEWGDYDGFDY Hv3-23 (SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 59)Sh24C05 GFTFSDY SDGGTY EWGDYDGFDY Hv3-30 (SEQ ID NO: 75) (SEQ ID NO: 76)(SEQ ID NO: 59) Hu24C05 GFTFSDY SDGGTY EWGDYDGFDY HvA (SEQ ID NO: 75)(SEQ ID NO: 76) (SEQ ID NO: 59) IMGT 24C05 GFTFSDYA ISDGGTYTAREWGDYDGFDY (SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 96) Sh24C05GFTFSDYA ISDGGTYT AREWGDYDGFDY Hv3-7 (SEQ ID NO: 94) (SEQ ID NO: 95)(SEQ ID NO: 96) Sh24C05 GFTFSDYA ISDGGTYT AREWGDYDGFDY Hv3-11(SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 96) Sh24C05 GFTFSDYAISDGGTYT AREWGDYDGFDY Hv3-11 (SEQ ID NO: 94) (SEQ ID NO: 95)(SEQ ID NO: 96) N62S Sh24C05 GFTFSDYA ISDGGTYT AREWGDYDGFDY Hv3-21(SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 96) Sh24C05 GFTFSDYAISDGGTYT AREWGDYDGFDY Hv3-23 (SEQ ID NO: 94) (SEQ ID NO: 95)(SEQ ID NO: 96) Sh24C05 GFTFSDYA ISDGGTYT AREWGDYDGFDY Hv3-30(SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 96) Hu24C05 GFTFSDYAISDGGTYT AREWGDYDGFDY HvA (SEQ ID NO: 94) (SEQ ID NO: 95)(SEQ ID NO: 96)

Humanized monoclonal antibody Kappa light chain CDR sequences (Kabat,Chothia, and IMGT definitions) are shown in Table 12.

TABLE 12 CDR1 CDR2 CDR3 Kabat/Chothia 24C05 RASQEISGYLS AASTLDSLQYDSYPYT (SEQ ID NO: 60) (SEQ ID NO: 61) (SEQ ID NO: 62) Sh24C05RASQEISGYLS AASTLDS LQYDSYPYT Kv1-9 (SEQ ID NO: 60) (SEQ ID NO: 61)(SEQ ID NO: 62) Sh24C05 RASQEISGYLS AASTLDS LQYDSYPYT Kv1-16(SEQ ID NO: 60) (SEQ ID NO: 61) (SEQ ID NO: 62) Sh24C05 RASQEISGYLSAASTLDS LQYDSYPYT Kv1-17 (SEQ ID NO: 60) (SEQ ID NO: 61) (SEQ ID NO: 62)Sh24C05 RASQEISGYLS AASTLDS LQYDSYPYT Kv1-33 (SEQ ID NO: 60)(SEQ ID NO: 61) (SEQ ID NO: 62) Sh24C05 RASQEISGYLS AASTLDS LQYDSYPYTKv1-39 (SEQ ID NO: 60) (SEQ ID NO: 61) (SEQ ID NO: 62) Hu24C05RASQEISGYLS AASTLDS LQYDSYPYT KvA (SEQ ID NO: 60) (SEQ ID NO: 61)(SEQ ID NO: 62) IMGT 24C05 QEISGY AAS LQYDSYPYT (SEQ ID NO: 101)(SEQ ID NO: 62) Sh24C05 QEISGY AAS LQYDSYPYT Kv1-9 (SEQ ID NO: 101)(SEQ ID NO: 62) Sh24C05 QEISGY AAS LQYDSYPYT Kv1-16 (SEQ ID NO: 101)(SEQ ID NO: 62) Sh24C05 QEISGY AAS LQYDSYPYT Kv1-17 (SEQ ID NO: 101)(SEQ ID NO: 62) Sh24C05 QEISGY AAS LQYDSYPYT Kv1-33 (SEQ ID NO: 101)(SEQ ID NO: 62) Sh24C05 QEISGY AAS LQYDSYPYT Kv1-39 (SEQ ID NO: 101)(SEQ ID NO: 62) Hu24C05 QEISGY AAS LQYDSYPYT KvA (SEQ ID NO: 101)(SEQ ID NO: 62)

In Tables 11 and 12, the longest CDR sequences for the immunoglobulinheavy chain and light chain are shown in bold.

To create the complete chimeric and humanized heavy or kappa chainantibody sequences, each variable sequence above is combined with itsrespective human constant region. For example, a complete heavy chaincomprises a heavy variable sequence followed by a human IgG1 heavy chainconstant sequence or a human IgG2 heavy chain constant sequence. Acomplete kappa chain comprises a kappa variable sequence followed by thehuman kappa light chain constant sequence.

Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain ConstantRegion (SEQ ID NO: 175)

1 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 61ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 121tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 181ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 241tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 301aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 361cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 421gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 481tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 541agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 601gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 661aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 721atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 781gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 841ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 901cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 961cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Human IgG Heavy Chain Constant Region (SEQID NO: 176)

1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 121psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 181styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 241mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Human IgG2 Heavy Chain ConstantRegion (SEQ ID NO: 177)

1 gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 61agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 121tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 181ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 241tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 301aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 361ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 421gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 481gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 541gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 601aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 661cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 721caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 781gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 841ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 901gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 961tccctgtctc cgggtaaa

Protein Sequence Defining the Human IgG2 Heavy Chain Constant Region(SEQ ID NO: 178)

1 astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61glyslssvvt vpssnfgtqt ytcnvdhkps ntkvdktver kccvecppcp appvagpsvf 121lfppkpkdtl misrtpevtc vvvdvshedp evqfnwyvdg vevhnaktkp reeqfnstfr 181vvsvltvvhq dwlngkeykc kvsnkglpap iektisktkg qprepqvytl ppsreemtkn 241qvsltclvkg fypsdiavew esngqpenny kttppmldsd gsfflysklt vdksrwqqgn 301vfscsvmhea lhnhytqksl slspgk

Nucleic Acid Sequence Encoding the Human Kappa Light Chain ConstantRegion (SEQ ID NO: 179)

1 cgcacagttg ctgcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc 61ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag 121tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac 181agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 241aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag 301tccttcaata ggggcgaatg t

Protein Sequence Defining the Human Kappa Light Chain Constant Region(SEQ ID NO: 180)

1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd 61skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

The following sequences represent the actual or contemplated full lengthheavy and light chain sequences (i.e., containing both the variable andconstant regions sequences) for each antibody described in this Example.Signal sequences for proper secretion of the antibodies are alsoincluded at the 5′ end of the DNA sequences or the amino terminal end ofthe protein sequences. It is also contemplated herein that the variableregion sequences can be ligated to other constant region sequences toproduce active full length IgG heavy and light chains.

Nucleic Acid Sequence Encoding the Full Length Chimeric 24C05 HeavyChain (Mouse Heavy Chain Variable Region and Human IgG1 Constant Region)(SEQ ID NO: 181)

1 atgaacttcg ggctcagctt gatgttcctt gtccttgtct taaaaggtgt ccagtgtgag 61gtgcagctgg tggaatctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc 121tgtgcagcct ctggattcac tttcagtgac tatgccatgt cttgggttcg ccagactccg 181gaaaagaggc tggagtgggt cgcaaccatt agtgatggtg gtacttacac ctactatcca 241gacaatgtaa agggccgatt caccatctcc agagacaatg ccaagaacaa cctgtacctg 301caaatgagcc atctgaagtc tgaggacaca gccatgtatt actgtgcaag agaatggggt 361gattacgacg gatttgacta ctggggccaa ggcaccactc tcacagtctc ctcggcctca 421acaaaaggac caagtgtgtt cccactcgcc cctagcagca agagtacatc cgggggcact 481gcagcactcg gctgcctcgt caaggattat tttccagagc cagtaaccgt gagctggaac 541agtggagcac tcacttctgg tgtccatact tttcctgctg tcctgcaaag ctctggcctg 601tactcactca gctccgtcgt gaccgtgcca tcttcatctc tgggcactca gacctacatc 661tgtaatgtaa accacaagcc tagcaatact aaggtcgata agcgggtgga acccaagagc 721tgcgacaaga ctcacacttg tcccccatgc cctgcccctg aacttctggg cggtcccagc 781gtctttttgt tcccaccaaa gcctaaagat actctgatga taagtagaac acccgaggtg 841acatgtgttg ttgtagacgt ttcccacgag gacccagagg ttaagttcaa ctggtacgtt 901gatggagtcg aagtacataa tgctaagacc aagcctagag aggagcagta taatagtaca 961taccgtgtag tcagtgttct cacagtgctg caccaagact ggctcaacgg caaagaatac 1021aaatgcaaag tgtccaacaa agcactccca gcccctatcg agaagactat tagtaaggca 1081aaggggcagc ctcgtgaacc acaggtgtac actctgccac ccagtagaga ggaaatgaca 1141aagaaccaag tctcattgac ctgcctggtg aaaggcttct accccagcga catcgccgtt 1201gagtgggaga gtaacggtca gcctgagaac aattacaaga caaccccccc agtgctggat 1261agtgacgggt ctttctttct gtacagtaag ctgactgtgg acaagtcccg ctggcagcag 1321ggtaacgtct tcagctgttc cgtgatgcac gaggcattgc acaaccacta cacccagaag 1381tcactgagcc tgagcccagg gaag

Protein Sequence Defining the Full Length Chimeric 24C05 Heavy Chain(Mouse Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQID NO: 182)

1 mnfglslmfl vlvlkgvqce vqlvesgggl vkpggslkls caasgftfsd yamswvrqtp 61ekrlewvati sdggtytyyp dnvkgrftis rdnaknnlyl qmshlksedt amyycarewg 121dydgfdywgq gttltvssas tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn 181sgaltsgvht fpavlqssgl yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks 241cdkthtcppc papellggps vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv 301dgvevhnakt kpreeqynst yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska 361kgqprepqvy tlppsreemt knqvsltclv kgfypsdiav ewesngqpen nykttppvld 421sdgsfflysk ltvdksrwqq gnvfscsvmh ealhnhytqk slslspgk

Nucleic Acid Sequence Encoding the Full Length Chimeric 24C05 LightChain (Mouse Kappa Chain Variable Region and Human Kappa ConstantRegion) (SEQ ID NO: 183)

1 atggacatga gggttcctgc tcacgttttt ggcttcttgt tgctctggtt tccaggtacc 61agatgtgaca tccagatgac ccagtctcca tcctccttat ctgcctctct gggagaaaga 121gtcagtctca cttgtcgggc aagtcaggaa attagtggtt acttaagctg gcttcagcag 181aaaccagatg gaactattaa acgcctgatc tacgccgcat ccactttaga ttctggtgtc 241ccaaaaaggt tcagtggcag taggtctggg tcagattatt ctctcaccat cggcagcctt 301gagtctgaag atcttgcaga ctattactgt ctacaatatg atagttatcc gtacacgttc 361ggagggggga ccaagctgga aataaaacgc acagtcgccg ctccctccgt gttcatcttt 421ccaccaagtg atgagcaact gaagtctggt actgcttcag tcgtgtgtct gctgaacaat 481ttctaccctc gagaagccaa agtccaatgg aaggtagaca acgcactgca gtccggcaat 541agccaagaat cagttaccga acaggattca aaggacagta catattccct gagcagcact 601ctgaccctgt caaaggccga ttacgagaaa cacaaggtct atgcttgcga agtgacacat 661cagggactgt ccagcccagt gacaaaatct tttaaccgtg gggagtgt

Protein Sequence Defining the Full Length Chimeric 24C05 Light Chain(Mouse Kappa Chain Variable Region and Human Kappa Constant Region) (SEQID NO: 184)

1 mdmrvpahvf gflllwfpgt rcdiqmtqsp sslsaslger vsltcrasqe isgylswlqq 61kpdgtikrli yaastldsgv pkrfsgsrsg sdysltigsl esedladyyc lqydsypytf 121gggtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

Nucleic Acid Sequence Encoding the Full Length Humanized sh24C05 Hv3-7Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO: 185)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgcgagg ttcagctggt ggaatctggc ggtgggcttg tacaaccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc 181caagcacccg ggaaaggact ggagtgggtt gccactatca gcgatggcgg aacgtatacc 241tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301ctctacctgc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-7 HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO: 186)

1 mdmrvpaqll gllllwlrga rcevqlvesg gglvqpggsl rlscaasgft fsdyamswvr 61qapgkglewv atisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-11Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO: 187)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgccaag ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg 181caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc 241tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-11 HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO: 188)

1 mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir 61qapgkglewv stisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-11N62S IgG1 Heavy Chain (Humanized Heavy Chain Variable Region and HumanIgG1 Constant Region) (SEQ ID NO: 189)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgccaag ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg 181caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc 241tattaccctg actccgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-11 N62SIgG1 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGConstant Region) (SEQ ID NO: 190)

1 mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir 61qapgkglewv stisdggtyt yypdsvkgrf tisrdnakns lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-11N62S IgG2 Heavy Chain (Humanized Heavy Chain Variable Region and HumanIgG2 Constant Region) (SEQ ID NO: 191)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgccaag ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg 181caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc 241tattaccctg actccgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcca ccaagggccc atcggtcttc cccctggcgc cctgctccag gagcacctcc 481gagagcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 541tcgtggaact caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcc 601tcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt cggcacccag 661acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gacagttgag 721cgcaaatgtt gtgtcgagtg cccaccgtgc ccagcaccac ctgtggcagg accgtcagtc 781ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcacg 841tgcgtggtgg tggacgtgag ccacgaagac cccgaggtcc agttcaactg gtacgtggac 901ggcgtggagg tgcataatgc caagacaaag ccacgggagg agcagttcaa cagcacgttc 961cgtgtggtca gcgtcctcac cgttgtgcac caggactggc tgaacggcaa ggagtacaag 1021tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga aaaccatctc caaaaccaaa 1081gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1141aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1201tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccat gctggactcc 1261gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1321aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1381ctctccctgt ctccgggtaa a

Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-11 N62SIgG2 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG2Constant Region) (SEQ ID NO: 192)

1 mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir 61qapgkglewv stisdggtyt yypdsvkgrf tisrdnakns lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapcsrsts estaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssnfgtq tytcnvdhkp sntkvdktve 241rkccvecppc pappvagpsv flfppkpkdt lmisrtpevt cvvvdvshed pevqfnwyvd 301gvevhnaktk preeqfnstf rvvsvltvvh qdwlngkeyk ckvsnkglpa piektisktk 361gqprepqvyt lppsreemtk nqvsltclvk gfypsdiave wesngqpenn ykttppmlds 421dgsfflyskl tvdksrwqqg nvfscsvmhe alhnhytqks lslspgk

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-21Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO: 193)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgcgagg ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc 181caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc 241tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301ctctatttgc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized 24C05 Hv3-21 HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO: 194)

1 mdmrvpaqll gllllwlrga rcevqlvesg gglvkpggsl rlscaasgft fsdyamswvr 61qapgkglewv stisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-23Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGConstant Region) (SEQ ID NO: 195)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgcgagg ttcagcttct ggaatctggc ggtgggcttg tacagccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc 181caagcacccg ggaaaggact ggagtgggtt tcaactatca gcgatggcgg aacgtatacc 241tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacag caagaacaca 301ctctatctcc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-23 HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO: 196)

1 mdmrvpaqll gllllwlrga rcevqllesg gglvqpggsl rlscaasgft fsdyamswvr 61qapgkglewv stisdggtyt yypdnvkgrf tisrdnsknt lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-30Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO: 197)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgccagg ttcagctggt ggaatctggc ggtggggtag tacaaccagg acggtccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc 181caagcacccg ggaaaggact ggagtgggtt gccactatca gcgatggcgg aacgtatacc 241tattaccctg acaatgtgaa gggtcggttc accatttcca gggataactc aaagaacacc 301ctctatctcc aaatgagtag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized of Sh24C05 Hv3-30Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO: 198)

1 mdmrvpaqll gllllwlrga rcqvqlvesg ggvvqpgrsl rlscaasgft fsdyamswvr 61qapgkglewv atisdggtyt yypdnvkgrf tisrdnsknt lylqmsslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Hu24C05 HvAHeavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO: 199)

1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61aggtgcgagg ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc 121agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc 181caagcacccg ggaaaggact ggagtgggtt gccactatca gcgatggcgg aacgtatacc 241tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga 361gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt 421tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc 481gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg 541agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc 601tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag 661acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa 721cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc 781ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca 841cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac 901tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat 961aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc 1021aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt 1081agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag 1141gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac 1201atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca 1261gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc 1321tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381acccagaagt cactgagcct gagcccaggg aag

Protein Sequence Defining the Full Length Humanized Hu24C05 HvA HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO: 200)

1 mdmrvpaqll gllllwlrga rcevqlvesg gglvkpggsl rlscaasgft fsdyamswvr 61qapgkglewv atisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti 361skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-9Light Chain (Humanized Kappa Chain Variable Region and Human ConstantRegion) (SEQ ID NO: 201)

1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61cgttgcgata ttcagttgac ccaatcacct agcttcctct cagcttccgt gggcgacaga 121gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtaccaacag 181aagcccggaa aagcccctaa gctgttgatc tatgctgcgt caaccttgga tagcggtgtc 241ccgagtcgat tctccggttc tggctccgga acagagttca ctctgacaat ttctagcctt 301cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt 361gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc 421ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac 481ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac 541agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc 601ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac 661cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt

Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-9 LightChain (Humanized Kappa Chain Variable Region and Human Constant Region)(SEQ ID NO: 202)

1 mdmrvpaqll gllllwlrga rcdiqltqsp sflsasvgdr vtitcrasqe isgylswyqq 61kpgkapklli yaastldsgv psrfsgsgsg teftltissl qpedfatyyc lqydsypytf 121gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-16Light Chain (Humanized Kappa Chain Variable Region and Human ConstantRegion) (SEQ ID NO: 203)

1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga 121gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtttcaacag 181aagcccggaa aggccccgaa gagcttgatc tatgctgcgt caaccttgga tagcggtgtc 241ccgagtcgat tctccggttc tggctccgga accgacttta ctctgacaat ttctagcctt 301cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt 361gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc 421ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac 481ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac 541agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc 601ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac 661cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt

Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-16 LightChain (Humanized Kappa Chain Variable Region and Human Constant Region)(SEQ ID NO: 204)

1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswfqq 61kpgkapksli yaastldsgv psrfsgsgsg tdftltissl qpedfatyyc lqydsypytf 121gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-17Light Chain (Humanized Kappa Chain Variable Region and Human ConstantRegion) (SEQ ID NO: 205)

1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga 121gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtatcaacag 181aagcccggaa aagccccaaa gaggttgatc tatgctgcgt caaccttgga tagcggtgtc 241ccgagtcgat tctccggttc tggctccgga accgagttca ctctgacaat ttctagcctt 301cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt 361gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc 421ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac 481ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac 541agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc 601ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac 661cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt

Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-17 LightChain (Humanized Kappa Chain Variable Region and Human Constant Region)(SEQ ID NO: 206)

1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq 61kpgkapkrli yaastldsgv psrfsgsgsg teftltissl qpedfatyyc lqydsypytf 121gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

Nucleic Acid Sequence Encoding the Full Length Humanized sh24C05 Kv1-33Light Chain (Humanized Kappa Chain Variable Region and Human ConstantRegion) (SEQ ID NO: 207)

1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga 121gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtaccaacag 181aagcccggaa aggcccccaa gctgttgatc tatgctgcgt caaccttgga tagcggtgtc 241ccgagtcgat tctccggttc tggctccgga acagacttta cttttacaat ttctagcctt 301cagccagagg acatcgccac gtactattgc ctccagtacg acagctatcc ctatacattt 361gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc 421ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac 481ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac 541agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc 601ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac 661cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt

Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-33 LightChain (Humanized Kappa Chain Variable Region and Human Constant Region)(SEQ ID NO: 208)

1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq 61kpgkapklli yaastldsgv psrfsgsgsg tdftftissl qpediatyyc lqydsypytf 121gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-39Light Chain (Humanized Kappa Chain Variable Region and Human ConstantRegion) (SEQ ID NO: 209)

1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga 121gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtatcaacag 181aagcccggaa aagcccctaa gctgttgatc tatgctgcgt caaccttgga tagcggtgtc 241ccgagtcgat tctccggttc tggctccgga actgacttca ctctgacaat ttctagcctt 301cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt 361gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc 421ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac 481ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac 541agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc 601ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac 661cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt

Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-39 LightChain (Humanized Kappa Chain Variable Region and Human Constant Region)(SEQ ID NO: 210)

1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq 61kpgkapklli yaastldsgv psrfsgsgsg tdftltissl qpedfatyyc lqydsypytf 121gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

Nucleic Acid Sequence Encoding the Full Length Humanized Hu24C05 KvALight Chain (Humanized Kappa Chain Variable Region and Human ConstantRegion) (SEQ ID NO: 211)

1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga 121gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gctgcaacag 181aagcccggag gcgccatcaa gaggttgatc tatgctgcgt caaccttgga tagcggtgtc 241ccgagtcgat tctccggttc tggctccgga agtgactaca ctctgacaat ttctagcctt 301cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt 361gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc 421ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac 481ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac 541agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc 601ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac 661cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt

Protein Sequence Defining the Full Length Humanized Hu24C05 KvA LightChain (Humanized Kappa Chain Variable Region and Human Constant Region)(SEQ ID NO: 212)

1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswlqq 61kpggaikrli yaastldsgv psrfsgsgsg sdytltissl qpedfatyyc lqydsypytf 121gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn 181sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec

For convenience, Table 13 provides a concordance chart showing the SEQID NO. of each sequence discussed in this Example.

TABLE 13 SEQ ID NO. Nucleic Acid or Protein 175 Human IgG1 constant -nucleic acid 176 Human IgG1 constant - protein 177 Human IgG2 constant -nucleic acid 178 Human IgG2 constant - protein 179 Human Kappaconstant - nucleic acid 180 Human Kappa constant - protein 181 Chimeric24C05 Mouse Heavy Chain Variable + Human IgG1 constant - nucleic acid182 Chimeric 24C05 Mouse Heavy Chain Variable + Human IgG1 constant -protein 183 Chimeric 24C05 Mouse Light Chain Variable + Human Kappaconstant - nucleic acid 184 Chimeric 24C05 Mouse Light Chain Variable +Human Kappa constant - protein 185 Humanized Sh24C05 Hv3-7 Heavy HumanVariable + Human IgG1 constant - nucleic acid 186 Humanized Sh24C05Hv3-7 Heavy Human Variable + Human IgG1 constant - protein 187 HumanizedSh24C05 Hv3-11 Heavy Human Variable + Human IgG1 constant - nucleic acid188 Humanized Sh24C05 Hv3-11 Heavy Human Variable + Human IgG1constant - protein 189 Humanized Sh24C05 Hv3-11 N62S IgG1 Heavy HumanVariable + Human IgG1 constant - nucleic acid 190 Humanized Sh24C05Hv3-11 N62S IgG1 Heavy Human Variable + Human IgG1 constant - protein191 Humanized Sh24C05 Hv3-11 N62S IgG2 Heavy Human Variable + Human IgG2constant - nucleic acid 192 Humanized Sh24C05 Hv3-11 N62S IgG2 HeavyHuman Variable + Human IgG2 constant - protein 193 Humanized Sh24C05Hv3-21 Heavy Human Variable + Human IgG1 constant - nucleic acid 194Humanized Sh24C05 Hv3-21 Heavy Human Variable + Human IgG1 constant -protein 195 Humanized Sh24C05 Hv3-23 Heavy Human Variable + Human IgG1constant - nucleic acid 196 Humanized Sh24C05 Hv3-23 Heavy HumanVariable + Human IgG1 constant - protein 197 Humanized Sh24C05 Hv3-30Heavy Human Variable + Human IgG1 constant - nucleic acid 198 HumanizedSh24C05 Hv3-30 Heavy Human Variable + Human IgG1 constant - protein 199Humanized Hu24C05 HvA Heavy Human Variable + Human IgG1 constant -nucleic acid 200 Humanized Hu24C05 HvA Heavy Human Variable + Human IgG1constant - protein 201 Humanized Sh24C05 Kv1-9 Human Variable + HumanKappa constant - nucleic acid 202 Humanized Sh24C05 Kv1-9 HumanVariable + Human Kappa constant - protein 203 Humanized Sh24C05 Kv1-16Human Variable + Human Kappa constant - nucleic acid 204 HumanizedSh24C05 Kv1-16 Human Variable + Human Kappa constant - protein 205Humanized Sh24C05 Kv1-17 Human Variable + Human Kappa constant - nucleicacid 206 Humanized Sh24C05 Kv1-17 Human Variable + Human Kappaconstant - protein 207 Humanized Sh24C05 Kv1-33 Human Variable + HumanKappa constant - nucleic acid 208 Humanized Sh24C05 Kv1-33 HumanVariable + Human Kappa constant - protein 209 Humanized Sh24C05 Kv1-39Human Variable + Human Kappa constant - nucleic acid 210 HumanizedSh24C05 Kv1-39 Human Variable + Human Kappa constant - protein 211Humanized Hu24C05 KvA Human Variable + Human Kappa constant - nucleicacid 212 Humanized Hu24C05 KvA Human Variable + Human Kappa constant -protein

Table 14 below shows antibodies containing chimeric immunoglobulin heavyand light chains and each of the possible combinations of thefull-length humanized immunoglobulin heavy and light chains.

TABLE 14 Antibody Name Light Chain Heavy Chain Sh24C05-1 24C05 ChimericKappa GP203 24C05 Chimeric Heavy IgG1 (SEQ ID NO: 184) (SEQ ID NO: 182)Sh24C05-14 Hu24C05 KvA Kappa Hu24C05 HvA IgG1 (SEQ ID NO: 212) (SEQ IDNO: 200) Sh24C05-15 Hu24C05 KvA Kappa Sh24C05 Hv3-21 Heavy IgG1 (SEQ IDNO: 212) (SEQ ID NO: 194) Sh24C05-16 Hu24C05 KvA Kappa Sh24C05 Hv3-23Heavy IgG1 (SEQ ID NO: 212) (SEQ ID NO: 196) Sh24C05-17 Hu24C05 KvAKappa Sh24C05 Hv3-30 Heavy IgG1 (SEQ ID NO: 212) (SEQ ID NO: 198)Sh24C05-18 Hu24C05 KvA Kappa Sh24C05 Hv3-7 Heavy IgG1 (SEQ ID NO: 212)(SEQ ID NO: 186) Sh24C05-19 Hu24C05 KvA Kappa Sh24C05 Hv3-11 Heavy IgG1(SEQ ID NO: 212) (SEQ ID NO: 188) Sh24C05-19 N62S Hu24C05 KvA KappaSh24C05 Hv3-11 N62S Heavy IgG1 IgG1 (SEQ ID NO: 212) (SEQ ID NO: 190)Sh24C05-19 N62S Hu24C05 KvA Kappa Sh24C05 Hv3-11 N62S Heavy IgG2 IgG2(SEQ ID NO: 212) (SEQ ID NO: 192) Sh24C05-20 Sh24C05 Kv1-16 KappaHu24C05 HvA IgG1 (SEQ ID NO: 204) (SEQ ID NO: 200) Sh24C05-21 Sh24C05Kv1-16 Kappa Sh24C05 Hv3-21 Heavy IgG1 (SEQ ID NO: 204) (SEQ ID NO: 194)Sh24C05-22 Sh24C05 Kv1-16 Kappa Sh24C05 Hv3-23 Heavy IgG1 (SEQ ID NO:204) (SEQ ID NO: 196) Sh24C05-23 Sh24C05 Kv1-16 Kappa Sh24C05 Hv3-30Heavy IgG1 (SEQ ID NO: 204) (SEQ ID NO: 198) Sh24C05-24 Sh24C05 Kv1-16Kappa Sh24C05 Hv3-7 Heavy IgG1 (SEQ ID NO: 204) (SEQ ID NO: 186)Sh24C05-25 Sh24C05 Kv1-16 Kappa Sh24C05 Hv3-11 Heavy IgG1 (SEQ ID NO:204) (SEQ ID NO: 188) Sh24C05-25 N62S Sh24C05 Kv1-16 Kappa Sh24C05Hv3-11 N62S Heavy IgG1 IgG1 (SEQ ID NO: 204) (SEQ ID NO: 190) Sh24C05-25N62S Sh24C05 Kv1-16 Kappa Sh24C05 Hv3-11 N62S Heavy IgG2 IgG2 (SEQ IDNO: 204) (SEQ ID NO: 192) Sh24C05-26 Sh24C05 Kv1-17 Kappa Hu24C05 HvAIgG1 (SEQ ID NO: 206) (SEQ ID NO: 200) Sh24C05-27 Sh24C05 Kv1-17 KappaSh24C05 Hv3-21 Heavy IgG1 (SEQ ID NO: 206) (SEQ ID NO: 194) Sh24C05-28Sh24C05 Kv1-17 Kappa Sh24C05 Hv3-23 Heavy IgG1 (SEQ ID NO: 206) (SEQ IDNO: 196) Sh24C05-29 Sh24C05 Kv1-17 Kappa Sh24C05 Hv3-30 Heavy IgG1 (SEQID NO: 206) (SEQ ID NO: 198) Sh24C05-30 Sh24C05 Kv1-17 Kappa Sh24C05Hv3-7 Heavy IgG1 (SEQ ID NO: 206) (SEQ ID NO: 186) Sh24C05-31 Sh24C05Kv1-17 Kappa Sh24C05 Hv3-11 Heavy IgG1 (SEQ ID NO: 206) (SEQ ID NO: 188)Sh24C05-31 N62S Sh24C05 Kv1-17 Kappa Sh24C05 Hv3-11 N62S Heavy IgG1 IgG1(SEQ ID NO: 206) (SEQ ID NO: 190) Sh24C05-31 N62S Sh24C05 Kv1-17 KappaSh24C05 Hv3-11 N62S Heavy IgG2 IgG2 (SEQ ID NO: 206) (SEQ ID NO: 192)Sh24C05-32 Sh24C05 Kv1-33 Kappa Hu24C05 HvA IgG1 (SEQ ID NO: 208) (SEQID NO: 200) Sh24C05-33 Sh24C05 Kv1-33 Kappa Sh24C05 Hv3-21 Heavy IgG1(SEQ ID NO: 208) (SEQ ID NO: 194) Sh24C05-34 Sh24C05 Kv1-33 KappaSh24C05 Hv3-23 Heavy IgG1 (SEQ ID NO: 208) (SEQ ID NO: 196) Sh24C05-35Sh24C05 Kv1-33 Kappa Sh24C05 Hv3-30 Heavy IgG1 (SEQ ID NO: 208) (SEQ IDNO: 198) Sh24C05-36 Sh24C05 Kv1-33 Kappa Sh24C05 Hv3-7 Heavy IgG1 (SEQID NO: 208) (SEQ ID NO: 186) Sh24C05-37 Sh24C05 Kv1-33 Kappa Sh24C05Hv3-11 Heavy IgG1 (SEQ ID NO: 208) (SEQ ID NO: 188) Sh24C05-37 N62SSh24C05 Kv1-33 Kappa Sh24C05 Hv3-11 N62S Heavy IgG1 IgG1 (SEQ ID NO:208) (SEQ ID NO: 190) Sh24C05-37 N62S Sh24C05 Kv1-33 Kappa Sh24C05Hv3-11 N62S Heavy IgG2 IgG2 (SEQ ID NO: 208) (SEQ ID NO: 192) Sh24C05-38Sh24C05 Kv1-9 Kappa Hu24C05 HvA IgG1 (SEQ ID NO: 202) (SEQ ID NO: 200)Sh24C05-39 Sh24C05 Kv1-9 Kappa Sh24C05 Hv3-21 Heavy IgG1 (SEQ ID NO:202) (SEQ ID NO: 194) Sh24C05-40 Sh24C05 Kv1-9 Kappa Sh24C05 Hv3-23Heavy IgG1 (SEQ ID NO: 202) (SEQ ID NO: 196) Sh24C05-41 Sh24C05 Kv1-9Kappa Sh24C05 Hv3-30 Heavy IgG1 (SEQ ID NO: 202) (SEQ ID NO: 198)Sh24C05-42 Sh24C05 Kv1-9 Kappa Sh24C05 Hv3-7 Heavy IgG1 (SEQ ID NO: 202)(SEQ ID NO: 186) Sh24C05-43 Sh24C05 Kv1-9 Kappa Sh24C05 Hv3-11 HeavyIgG1 (SEQ ID NO: 202) (SEQ ID NO: 188) Sh24C05-43 N62S Sh24C05 Kv1-9Kappa Sh24C05 Hv3-11 N62S Heavy IgG1 IgG1 (SEQ ID NO: 202) (SEQ ID NO:190) Sh24C05-43 N62S Sh24C05 Kv1-9 Kappa Sh24C05 Hv3-11 N62S Heavy IgG2IgG2 (SEQ ID NO: 202) (SEQ ID NO: 192) Sh24C05-44 Sh24C05 Kv1-39 KappaHu24C05 HvA IgG1 (SEQ ID NO: 210) (SEQ ID NO: 200) Sh24C05-45 Sh24C05Kv1-39 Kappa Sh24C05 Hv3-21 Heavy IgG1 (SEQ ID NO: 210) (SEQ ID NO: 194)Sh24C05-46 Sh24C05 Kv1-39 Kappa Sh24C05 Hv3-23 Heavy IgG1 (SEQ ID NO:210) (SEQ ID NO: 196) Sh24C05-47 Sh24C05 Kv1-39 Kappa Sh24C05 Hv3-30Heavy IgG1 (SEQ ID NO: 210) (SEQ ID NO: 198) Sh24C05-48 Sh24C05 Kv1-39Kappa Sh24C05 Hv3-7 Heavy IgG1 (SEQ ID NO: 210) (SEQ ID NO: 186)Sh24C05-49 Sh24C05 Kv1-39 Kappa Sh24C05 Hv3-11 Heavy IgG1 (SEQ ID NO:210) (SEQ ID NO: 188) Sh24C05-49 N62S Sh24C05 Kv1-39 Kappa Sh24C05Hv3-11 N62S Heavy IgG1 IgG1 (SEQ ID NO: 210) (SEQ ID NO: 190) Sh24C05-49N62S Sh24C05 Kv1-39 Kappa Sh24C05 Hv3-11 N62S Heavy IgG2 IgG2 (SEQ IDNO: 210) (SEQ ID NO: 192)

The antibody construct containing the full length chimeric heavy andlight chains is designated below:

-   -   Chimeric 24C05=Full Length Chimeric 24C05 Heavy Chain (Mouse        Variable Region and Human IgG Constant Region) (SEQ ID NO: 182)        plus Full Length Chimeric 24C05 Light Chain (Mouse Variable        Region and Human Kappa Constant Region) (SEQ ID NO: 184)

Four of the possible antibody constructs containing the full lengthimmunoglobulin heavy and light chains containing humanized variableregions are designated below:

-   -   Sh24C05-25 N62S IgG1=Humanized Sh24C05 Hv3-11 N62S Heavy Chain        Variable Region and Human IgG Constant Region (SEQ ID NO: 190)        plus Sh24C05 Kv1-16 Light Chain Variable Region and Human Kappa        Constant Region (SEQ ID NO: 204)    -   Sh24C05-25 N62S IgG2=Humanized Sh24C05 Hv3-11 N62S Heavy Chain        Variable Region and Human IgG2 Constant Region (SEQ ID NO: 192)        plus Sh24C05 Kv1-16 Light Chain Variable Region and Human Kappa        Constant Region (SEQ ID NO: 204)    -   Sh24C05-31 N62S IgG1=Humanized Sh24C05 Hv3-11 N62S Heavy Chain        Variable Region and Human IgG Constant Region (SEQ ID NO: 190)        plus Sh24C05 Kv1-17 Light Chain Variable Region and Human Kappa        Constant Region (SEQ ID NO: 206)    -   Sh24C05-31 N62S IgG2=Humanized Sh24C05 Hv3-11 N62S Heavy Chain        Variable Region and Human IgG2 Constant Region (SEQ ID NO: 192)        plus Sh24C05 Kv1-17 Light Chain Variable Region and Human Kappa        Constant Region (SEQ ID NO: 206)

B. Binding Affinities of Humanized and Chimeric Anti-ErbB3 MonoclonalAntibodies

The binding affinities and kinetics of interaction of monoclonalantibodies produced in Example 12 against recombinant human ErbB3monomeric protein (cleaved rhErbB3) were measured by surface plasmonresonance using a BIACORE® T100 (Biacore) instrument. Monomeric ErbB3was obtained by protease cleavage of rhErbB3-Fc (R&D Systems, Cat. No.348-RB).

Goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog No. 109-005-098)was immobilized on carboxymethylated dextran CM4 sensor chips (Biacore,Catalog No. BR-1005-34) by amine coupling (Biacore, Catalog No.BR-1000-50) using a standard coupling protocol according to the vendor'sinstructions. The analyses were performed at 37° C. using PBS(Invitrogen, Catalog No. 14040-133) containing 0.05% surfactant P20(Biacore, Catalog No. BR-1000-54) as running buffer.

The antibodies were captured in individual flow cells at a flow rate of60 l/minute. Injection time was varied for each antibody to yield anR_(max) between 30 and 60 RU. Buffer or cleaved rhErbB3 diluted inrunning buffer was injected sequentially over a reference surface (noantibody captured) and the active surface (antibody to be tested) for300 seconds at 60 l/minute. The dissociation phase was monitored for upto 1200 seconds. The surface was then regenerated with two 60 secondinjections of Glycine pH 2.25 (made from Glycine pH 2.0 (Biacore,Catalog No. BR-1003-55) and pH 2.5 (Biacore, Catalog No. BR-1003-56)) at60 l/minute. For the initial screening, only one or two concentrationsof cleaved rhErbB3 were tested, typically 5.0 and 1.25 nM (results aresummarized in Table 15).

Kinetic parameters were determined using the kinetic function of theBIAEVALUATION™ software (Biacore) with double reference subtraction.Kinetic parameters for each antibody, k_(a) (association rate constant),k_(d) (dissociation rate constant) and K_(D) (equilibrium dissociationconstant) were determined. Kinetic values of the monoclonal antibodieson rhErbB3-Fc at 25° C. are summarized in Table 5.

TABLE 15 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Sh24C05-12.52E+06 4.48E−04 1.78E−10 3 Sh24C05-14 2.88E+06 4.98E−04 1.73E−10 2Sh24C05-15 2.67E+06 4.99E−04 1.87E−10 2 Sh24C05-16 2.75E+06 4.04E−041.47E−10 2 Sh24C05-17 2.79E+06 4.17E−04 1.50E−10 2 Sh24C05-18 2.88E+064.63E−04 1.61E−10 2 Sh24C05-19 3.00E+06 2.55E−04 8.55E−11 2 Sh24C05-202.67E+06 5.91E−04 2.21E−10 2 Sh24C05-21 3.11E+06 6.62E−04 2.20E−10 2Sh24C05-22 2.79E+06 6.01E−04 2.16E−10 2 Sh24C05-23 2.79E+06 7.21E−042.63E−10 2 Sh24C05-24 2.90E+06 6.28E−04 2.18E−10 2 Sh24C05-25 2.63E+064.59E−04 1.75E−10 2 Sh24C05-26 3.36E+06 7.39E−04 2.20E−10 2 Sh24C05-273.34E+06 7.98E−04 2.40E−10 2 Sh24C05-28 3.26E+06 6.14E−04 1.89E−10 2Sh24C05-29 3.25E+06 5.88E−04 1.82E−10 2 Sh24C05-30 4.48E+06 7.87E−041.90E−10 2 Sh24C05-31 3.47E+06 2.92E−04 8.65E−11 2 Sh24C05-32 9.98E+066.02E−03 6.03E−10 1 Sh24C05-33 4.02E+06 4.33E−03 1.08E−09 1 Sh24C05-341.09E+07 6.00E−03 5.52E−10 1 Sh24C05-35 8.44E+06 5.53E−03 6.55E−10 1Sh24C05-36 5.18E+06 4.34E−03 8.37E−10 1 Sh24C05-37 5.94E+06 2.00E−033.74E−10 2 Sh24C05-38 2.71E+07 1.54E−02 5.67E−10 1 Sh24C05-39 1.18E+079.67E−03 8.19E−10 1 Sh24C05-40 2.11E+07 1.06E−02 5.03E−10 1 Sh24C05-411.81E+07 1.21E−02 6.69E−10 1 Sh24C05-42 7.35E+06 6.82E−03 9.27E−10 1Sh24C05-43 6.16E+06 3.58E−03 5.82E−10 1 Sh24C05-44 7.96E+06 5.12E−036.44E−10 1 Sh24C05-45 8.57E+06 6.06E−03 7.07E−10 1 Sh24C05-46 7.99E+064.40E−03 5.51E−10 1 Sh24C05-47 7.98E+06 4.41E−03 5.53E−10 1 Sh24C05-488.72E+06 4.90E−03 5.62E−10 1 Sh24C05-49 4.08E+06 1.70E−03 4.16E−10 2

The results in Table 15 demonstrate that the chimeric and each of thehumanized 24C05 antibodies have fast association rates (k_(a)), veryslow disassociation rates (k_(d)) and very high affinities (K_(D)). Inparticular, the antibodies have affinities ranging from about 87 pM toabout 1 nM.

The binding affinities and kinetics of certain purified monoclonalantibodies were also determined. To further characterize certainantibodies, the surface plasmon resonance experiments described abovewere conducted using concentrations of cleaved rhErbB3 between 0.3125 nMand 5.0 nM (a 2-fold serial dilution).

The kinetic values of certain purified monoclonal antibodies (i.e.,Sh24C05-1, Sh24C05-25, Sh24C05-25 N62S IgG1, Sh24C05-25 N62S IgG2,Sh24C05-31, Sh24C05-31 N62S IgG1, and Sh24C05-31 N62S IgG2) on cleavedrhErbB3 at 37° C. are summarized in Table 16.

TABLE 16 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Sh24C05-1 3.5E+064.4E−04 1.4E−10 3 Sh24C05-25 4.0E+06 5.0E−04 1.3E−10 4 Sh24C05-25 N62S2.9E+06 4.5E−04 1.6E−10 4 IgG1 Sh24C05-25 N62S 2.7E+06 3.4E−04 1.2E−10 4IgG2 Sh24C05-31 4.7E+06 2.8E−04 6.3E−11 3 Sh24C05-31 N62S 3.5E+062.7E−04 7.6E−11 6 IgG1 Sh24C05-31 N62S 3.2E+06 2.4E−04 7.4E−11 3 IgG2

The results in Table 16 demonstrate the purified antibodies have a haveaffinities ranging from about 63 pM to about 160 pM when tested at 37°C.

C. Comparison of Other Anti-ErbB3 Antibodies

Three human antibodies that inhibit the function of human ErbB3 wereconstructed and expressed using published information. One antibody,referred to as Ab #6, was constructed as a human IgG2/Lambda antibodybased the disclosure of Schoeberl et al., US 2009/0291085 (MerrimackPharmaceuticals, Inc.). Two additional antibodies, referred to as U1-53and U1-59, were constructed as human IgG1/Kappa antibodies based on thedisclosure of Rothe et al., US 2008/0124345 (U3 Pharma AG and Amgen,Inc.).

Kinetic parameters for the Ab#6, U1-53, and U1-59 antibodies weredetermined by BIACORE® at 37° C. using cleaved rhErbB3 (monomer) asdescribed above (See Section B. Binding Affinities of Humanized andChimeric Anti-ErbB3 Monoclonal Antibodies). Both BIACORE® sensorgrams(FIG. 17) and kinetic values (Table 17) are displayed for each antibody.

TABLE 17 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Sh24C05-31 N62S  3.5+06 2.7E−04 7.6E−11 6 IgG1 Ab#6 9.3E+05 1.9E−04 2.3E−10 3 U1-591.8E+06 9.4E−04 5.3E−10 3 U1-53 — — — —

The results in Table 17 demonstrate that the overall equilibriumdissociation constant (K_(D)) for the Sh24C05-31 N62S IgG1 (76 pM) wassmaller (i.e., higher affinity) than the K_(D) for the Ab#6 and U1-59antibodies (230 pM (p<0.01) and 530 pM (p<0.0005), respectively). Theequilibrium dissociation constant (K_(D)) for U1-53 could not determinedbecause of poor curve fits (see FIG. 17, which shows a fast K_(off) rateof U1-53). The K_(D) of Ab #6, U1-53, and U1-59 antibodies can also becompared with other humanized 24C05 variants by comparing Tables 16 and17.

Therefore, the affinity for Sh24C05-31 N62S IgG1 is significantly higherthan the affinity of Ab#6 and U1-59 as disclosed herein.

Example 13—Neutralization Activity of the Humanized Anti-ErbB3Antibodies

In this example, the humanized antibodies produced in Example 12 weretested for their ability to inhibit rhErbB3 binding to NRG1-β1 by ECLassay. Multi-array 96-well standard binding plates (Meso ScaleDiscovery, Cat. No. L15XA-3) were coated with 50 μl of 0.5 μg/mLrhErbB3/Fc (R&D systems, Cat. No. 348-RB) in PBS (Invitrogen, Cat. No.14040-133) for one hour at room temperature with no agitation. Theplates then were washed three times with PBS+0.1% Tween20 (Sigma P5927)and blocked with 200 μl of 100% Horse Serum, heat inactivated (GIBCO,Cat. No. 26050-088) for 1.5 hours at room temperature. After washing theplates three times with PBS+0.1% Tween, 25 μl of the antibody dilutionswere added to the plates for another hour at room temperature withagitation. Ligand NRG1-β1 (R&D Systems, Cat. No. 377-HB, 26 kDa) wasadded to the wells at a final concentration of 0.25 μg/ml. The plateswere washed three times with PBS+0.1% Tween and incubated with 25 μl of1 μg/mL biotinylated antibody against human NRG1-β1 (R&D systems, Cat.No BAF377) preincubated for one hour with SULTO-TAG Streptavidin (MesoScale Discovery, Cat. No R32AD-5) for one hour at room temperature withagitation. The plates then were washed three times with PBS+0.1% Tween,and 150 μl of 1× read buffer (Meso Scale Discovery, Cat. No. R92TC-1)was added to each well before the plates were analyzed on a Sector®Imager 2400 (Meso Scale Discovery) instrument.

The interaction of NRG1-β1 with rhErbB3 was inhibited by antibodiesSh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, andSh24C05-31 N62S-IgG2 (FIG. 18A). The Ab#6 IgG2 antibody as described inSchoeberl et al. (supra) and the U1-53 and U1-59 antibodies as describedin Rothe et al. (supra) were also tested for their ability to inhibitErbB3 binding to NRG1-β1. As shown in FIG. 18B, each of the Ab#6 IgG2,U1-53, and U1-59 antibodies inhibited ErbB3 binding to NRG1-β1.

The IC₅₀ values for neutralization of NRG1-β1 binding to hErbB3 for thehumanized 24C05 antibodies (i.e., Sh24C05-25 N62S-IgG1, Sh24C05-25N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2) werecalculated and are summarized in Table 18. The IC₅₀ values for theNRG1-β1 neutralization activity of the anti-ErbB3 human antibodies Ab#6IgG2, U1-53 and U1-59 are also shown in Table 18.

TABLE 18 IC₅₀ (nM) Antibody Average Standard deviation n Sh24C05-25N62S-IgG1 0.1219 0.0173 4 Sh24C05-25 N62S-IgG2 0.1117 0.0154 4Sh24C05-31 N62S-IgG1 0.1242 0.0391 5 Sh24C05-31 N62S-IgG2 0.0860 0.05884 U1-53 0.1128 0.0615 3 U1-59 0.3181 0.0274 3 Ab#6 IgG2 1.5161 0.5883 5

The results in Table 18 demonstrate that antibodies Sh24C05-25N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31N62S-IgG2 efficiently neutralized NRG1-β1 binding to rhErbB3. While theanti-ErbB3 human antibodies Ab#6 IgG2, U1-53 and U1-59 also showedneutralization activity, the humanized Sh24C05 antibodies (i.e.,Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, andSh24C05-31 N62S-IgG2) had superior neutralization capacity than U1-59 orAb#6 IgG2.

Example 14—Anti-Proliferative Activity

In this example, the humanized antibodies produced in Example 12 weretested for their ability to inhibit NRG1-(1 dependent proliferation ofcells in the BaF/3 cell system engineered to express both human Her2 andErbB3.

BaF/3 cells expressing Her2 and ErbB3 receptors as described in Example6 were treated with anti-ErbB3 antibodies in the absence of WEHIconditioned media but in the presence of NRG1-β1 (100 ng/ml). Assayswere conducted in a 96-well plate (5,000 cells/well) in the presence ofNRG1-β 1 (100 ng/ml) and various concentrations of antibodies(0.018-5000 ng/ml in 100 μl of final volume). MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assayswere conducted 3-4 days post NRG1-β1 stimulation.

The results demonstrate that Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2,Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 inhibited NRG inducedHer2/ErbB3-BaF/3 cell proliferation in a dose dependent manner (FIG.19A).

The IC₅₀ values for the inhibition of NRG1-β1 dependent Her2/ErbB3-BaF/3cell line proliferation with the humanized 24C05 antibodies (i.e.,Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1,Sh24C05-31 N62S-IgG2) were calculated and are summarized in Table 19.

TABLE 19 Her2/ErbB3-BaF/3, NRG1-β1 Dependent Proliferation Antibody IC₅₀(nM)-Average Standard deviation n Sh24C05-25 N62S-IgG1 0.0981 0.0187 2Sh24C05-25 N62S-IgG2 0.2482 0.0124 2 Sh24C05-31 N62S-IgG1 0.1245 0.01815 Sh24C05-31 N62S-IgG2 0.2392 0.0217 2 U1-53 0.8128 0.0268 3 U1-590.8364 0.0434 5 Ab#6 IgG2 6.3015 0.8577 2

The results in Table 19 demonstrate that antibodies Sh24C05-25N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31N62S-IgG2 strongly inhibited NRG1-β1-induced proliferation of BaF/3cells expressing Her2/ErbB3.

The inhibitory activity of anti-ErbB3 Ab#6 IgG2, U1-53 and U1-59antibodies were also tested in the NRG1-β1 dependent Her2/ErbB3-BaF/3cells proliferation assay. As shown in FIG. 19B, the results demonstratethat the Ab#6 IgG2, U1-53 and U1-59 antibodies inhibited NRG inducedHer2/ErbB3-BaF/3 cell proliferation in a dose dependent manner.Inhibition data of NRG1-β1 dependent Her2/ErbB3-BaF/3 cell proliferationwith antibodies Ab#6 IgG2, U1-53 and U1-59 are summarized in Table 19.The results in Table 19 demonstrate that antibodies Ab#6 IgG2, U1-53,and U1-59 inhibited NRG1-β1-induced proliferation of Her2/ErbB3-BaF/3cells. A comparison of the inhibitory activity of the tested anti-ErbB3antibodies in the NRG1-β1 dependent Her2/ErbB3-BaF/3 cells proliferationassay indicates that the inhibitory activity of the humanized Sh24C05antibodies is superior to the inhibitory activity of the Ab#6 IgG2,U1-53 and U1-59 antibodies (e.g., the IC₅₀ was 0.1245 nM for Sh24C05-31N62S-IgG1 compared to 0.8128 nM for U1-53).

Example 15—Inhibition of Downstream Signaling in SKBR-3 Cells

This example describes a characterization of the humanized antibodiesproduced in Example 12 for their ability to degrade total ErbB3 andinhibit phosphorylation of ErbB3 in exponentially growing SKBR-3 cells.

The breast cancer SKBR-3 cells were maintained as recommended by ATCC.Cells maintained in full serum condition were treated for 1, 2, 4 or 6hours with 40 pg/ml of anti-ErbB3 antibody (i.e., Sh24C05-25 N62S-IgG1,Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2).Lysates were either analyzed by ELISA with the Total-ErbB3 and thePhospho-ErbB3 kit from R&D Systems (Cat. No DYC234 and Cat. No DYC 1769,respectively).

The results demonstrate that anti-ErbB3 antibodies Sh24C05-25 N62S-IgG1,Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2inhibit at least 50% of the phosphorylation of ErbB3 in exponentiallygrowing SKBR-3 cells (FIG. 20).

The results also demonstrate that anti-ErbB3 antibodies Sh24C05-25N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31N62S-IgG2 degraded at least 50% of the total ErbB3 receptor present inexponentially growing SKBR-3 cells (FIG. 21).

Example 16—Inhibition of BxPC3 Tumor Xenograft Growth

The ability of the humanized monoclonal antibodies produced in Example12 to inhibit tumor growth were tested in a BxPC3 pancreatic xenograftmodel. Human pancreatic BxPC3 cells were grown in culture in 37° C. inan atmosphere containing 5% CO2, using RMPI medium containing 10% fetalbovine serum. BxPC3 cells were inoculated subcutaneously into the flankof 8-week old female CB.17 SCID mice (Taconic Labs) with 10×10⁶ cellsper mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumormeasurements were taken twice weekly using vernier calipers. Tumorvolume was calculated using the formula: width×width×length/2. Whentumors reached approximately 200 mm³, the mice were randomized into 8groups of 10 mice each. One group received PBS, another received huIgGcontrol, and another received muIgG control. Each of the remaining fivegroups received one of the antibodies (i.e., murine 24C05, Sh24C05-25N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1 or Sh24C05-31N62S-IgG2). All of the antibodies were dosed at 2 mg/kg body weight,twice per week, by intra-peritoneal injection for 7 weeks. Tumor volumesand mouse body weights were recorded twice per week. Tumor growthinhibition was analyzed using ANOVA and is expressed as percentinhibition compared to the PBS control.

The tested humanized antibodies were active in vivo. All four humanizedanti-ErbB3 antibodies had similar efficacy in the BxPC3 model when dosedat 2 mg/kg, ranging from 75-80% tumor growth inhibition (p<0.001) (i.e.,Sh24C05-25 N62S-IgG1, 75%; Sh24C05-25 N62S-IgG2, 76%; Sh24C05-31N62S-IgG1, 79%; and Sh24C05-31 N62S-IgG2, 80%) at day 28 of the study(FIG. 22). The murine antibody demonstrated 65% tumor growth inhibitionin this study (p<0.05). These results suggest similar potency andactivity of the four humanized antibodies in this model.

The ability of the humanized monoclonal antibodies U1-53, U1-59, andAb#6 IgG2 to inhibit tumor growth were also tested in a BxPC3 xenograftmodel. Using the protocol described above, BxPC3 tumors were generatedin CB.17 SCID mice. When tumors reached approximately 200 mm³, the micewere randomized into 11 groups of 10 mice each. One group received PBSand another received huIgG control. Each of the other nine groupsreceived one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1,U1-53, U1-59, or Ab#6 IgG2). The antibodies were dosed either at 0.5mg/kg, 1 mg/kg, or 5 mg/kg body weight, twice per week, byintra-peritoneal injection for 7 weeks. Tumor volumes and mouse bodyweights were recorded twice per week. Tumor growth inhibition wasanalyzed using ANOVA and is expressed as percent inhibition compared tothe PBS control.

Tumor growth inhibition data determined at day 29 following treatmentwith one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59,or Ab#6 IgG2) is shown in Table 20.

TABLE 20 Tumor ANOVA ANOVA Growth Analysis Analysis Treatment Inhibition(Compared (Compared Gr. Agent mg/kg (%) to PBS) to hIgG) 1 PBS — NA NANA 2 hIgG 5 29.2 NS NS 3 Sh24C05-31 0.5 63.3 P < 0.001 P < 0.01 N62S-IgG1 4 Sh24C05-31 1 75.0 P < 0.001 P < 0.001 N62S-IgG1 5 Sh24C05-315 76.5 P < 0.001 P < 0.001 N62S-IgG1 6 Ab#6 IgG2 0.5 31.5 P < 0.05  NS 7Ab#6 IgG2 1 2.1 NS NS 8 Ab#6 IgG2 5 40.6 P < 0.001 NS 9 U1-59 0.5 32.6 P< 0.01  NS 10 U1-59 1 52.9 P < 0.001 NS 11 U1-59 5 60.3 P < 0.001 P <0.05 

The results demonstrate that Sh24C05-31 N62S-IgG1 showed the greatesttumor growth inhibition by day 29 (76.5%, p<0.001) at a dose of 5 mg/kgin the BxPC3 pancreatic xenograft model. The U 1-59 and Ab#6 IgG2antibodies demonstrated approximately 60% and 41% tumor growthinhibition at a dose of 5 mg/kg in the BxPC3 model, respectively(P<0.001).

The results also demonstrate that Sh24C05-31 N62S-IgG1 showed thegreatest tumor growth inhibition by day 29 at a dose of 0.5 mg/kg(63.3%, p<0.001) and at a dose of 1 mg/kg (75.0%, p<0.001) in the BxPC3pancreatic xenograft model. The U1-59 and AB#6 IgG2 antibodiesdemonstrate approximately 33% (p<0.01) and 31% (p<0.05) tumor growthinhibition at a dose of 0.5 mg/kg in the BxPC3 model, respectively. TheU1-59 and AB#6 IgG2 antibodies demonstrated approximately 53% (p<0.001)and 2% (not significant) tumor growth inhibition at a dose of 1.0 mg/kgin the BxPC3 model, respectively.

Example 17—Inhibition of Calu-3 Tumor Xenograft Growth

The ability of the humanized monoclonal antibodies produced in Example12 to inhibit tumor growth was tested in a Calu-3 non-small cell lungcancer xenograft model. The ability of the humanized monoclonalantibodies U1-59 and Ab#6 IgG2, as described in Example 12, to inhibittumor growth were also tested in the same model.

Human Non-Small Cell Lung Cancer Calu-3 cells were grown in culture in37° C. in an atmosphere containing 5% CO2, using EMEM medium containing10% fetal bovine serum. Calu-3 cells were inoculated subcutaneously intothe flank of 8-week old female NCR nude mice (Taconic Labs) with 10×10⁶cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumormeasurements were taken twice weekly using vernier calipers. Tumorvolume was calculated using the formula: width×width×length/2.

When tumors reached approximately 200 mm³, the mice were randomized into11 groups of 10 mice each. One group received PBS and another receivedmuIgG control. Each of the other nine groups received one of thehumanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2)at a dose of either 5 mg/kg, 10 mg/kg or 20 mg/kg body weight, twice perweek, by intra-peritoneal injection for 4 weeks. Tumor volumes and mousebody weights were recorded twice per week. Tumor growth inhibition wasanalyzed using ANOVA and is expressed as percent inhibition compared tothe PBS control.

Tumor growth inhibition data determined at day 26 following treatmentwith one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59,or Ab#6 IgG2) is shown in Table 21.

TABLE 21 Tumor ANOVA ANOVA Growth Analysis Analysis Treatment Inhibition(Compared (Compared Gr. Agent mg/kg (%) to PBS) to hIgG) 1 PBS — NA NANA 2 muIgG 20 −1.2 NS NA 3 Sh24C05-31 5 62.3 P < 0.001 P < 0.001N62S-IgG1 4 Sh24C05-31 10 62.0 P < 0.001 P < 0.001 N62S-IgG1 5Sh24C05-31 20 69.0 P < 0.001 P < 0.001 N62S-IgG1 6 Ab#6 IgG2 5 24.7 NSNS 7 Ab#6 IgG2 10 35.9 P < 0.01  P < 0.01  8 Ab#6 IgG2 20 48.4 P < 0.001P < 0.001 9 U1-59 5 47.8 P < 0.001 P < 0.001 10 U1-59 10 56.7 P < 0.001P < 0.001 11 U1-59 20 57.7 P < 0.001 P < 0.001

The results using the Calu-3 non-small cell lung cancer xenograft modeldemonstrate that Sh24C05-31 N62S-IgG1 showed the greatest tumor growthinhibition by day 26 at all doses tested (i.e., 5 mg/kg, 10 mg/kg, and20 mg/kg of body weight).

For example, at the 10 mg/kg dose, Sh24C05-31 N62S-IgG1 showed thegreatest tumor growth inhibition by day 26 (62%, P<0.001) when comparedto Ab#6 IgG2 (36%, NS) or U1-59 (57%, P<0.001). At the 20 mg/kg dose,Sh24C05-31 N62S-IgG1 also showed the greatest tumor growth inhibition byday 26 (69%, P<0.001) when compared to Ab#6 IgG2 (48%, P<0.001) or U1-59(58%, P<0.001).

Example 18—Inhibition of MDA-MB-453 Tumor Xenograft Growth

The ability of the humanized monoclonal antibodies produced in Example12 to inhibit tumor growth were tested in a MDA-MB-453 breast xenograftmodel (which is a HER2 positive breast model). The ability of thehumanized monoclonal antibodies U1-59 and Ab#6 IgG2, as described inExample 12, to inhibit tumor growth were also tested in the same model.

Human Breast MDA-MB-453 cells were grown in culture in 37° C. in anatmosphere containing 0% CO2, using Leibovitz ATCC medium (Cat No.30-2008) containing 10% fetal bovine serum. MDA-MB-453 cells wereinoculated subcutaneously into the flank of 8-week old female NOD SCIDmice (Taconic Labs) with 20×10⁶ cells per mouse in 50% matrigel (BDBiosciences, Cat No. 356237). Tumor measurements were taken twice weeklyusing vernier calipers. Tumor volume was calculated using the formula:width×width×length/2.

When tumors reached approximately 200 mm³, the mice were randomized into7 groups of 10 mice each. One group received PBS and another receivedhuIgG control. Each of the other nine groups received one of thehumanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2).Sh24C05-31 N62S-IgG1 was dosed either at 5 mg/kg, 10 mg/kg, or 20 mg/kgbody weight, twice per week, by intra-peritoneal injection for more than10 weeks; U1-59, or Ab#6 were dosed at 10 mg/kg with the same frequency.Tumor volumes and mouse body weights were recorded twice per week. Tumorgrowth inhibition was analyzed using ANOVA and is expressed as percentinhibition compared to the PBS control.

Tumor growth inhibition data determined at day 71 following treatmentwith one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59,or Ab#6 IgG2) is shown in Table 22.

TABLE 22 Tumor ANOVA ANOVA Growth Analysis Analysis Treatment Inhibition(Compared (Compared Gr. Agent mg/kg (%) to PBS) to hIgG) 1 PBS — NA NANA 2 hIgG 20 28.87 p < 0.001 p < 0.001 3 Sh24C05-31 5 86.57 p < 0.001 p< 0.001 N62S-IgG1 4 Sh24C05-31 10 84.09 p < 0.001 p < 0.001 N62S-IgG1 5Sh24C05-31 20 85.26 p < 0.001 p < 0.001 N62S-IgG1 6 Ab#6 IgG2 10 62.48 p< 0.001 p < 0.001 7 U1-59 10 83.93 p < 0.001 p < 0.001

The results using the MDA-MB-453 xenograft model demonstrate thatSh24C05-31 N62S-IgG1 showed potent tumor growth inhibition by day 71 atall doses tested (i.e., 5 mg/kg, 10 mg/kg, and 20 mg/kg of body weight).

The results also demonstrate that at the 10 mg/kg dose, Sh24C05-31N62S-IgG1 showed greater tumor growth inhibition by day 71 (84%,P<0.001) when compared to Ab#6 IgG2 (62%, P<0.001). Sh24C05-31 N62S-IgG1showed equivalent tumor growth inhibition as U1-59 at the same dose.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andthe range of equivalency of the claims are intended to be embracedtherein.

What is claimed is:
 1. An isolated antibody that binds human ErbB3comprising (i) an immunoglobulin heavy chain variable region comprisinga CDR_(H1) comprising the amino acid sequence of SEQ ID NO: 5, aCDR_(H2) comprising the amino acid sequence of SEQ ID NO: 6, and aCDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7; and (ii) animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO: 8, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO: 9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:
 10. 2. The antibody ofclaim 1, wherein the CDR sequences are interposed between human orhumanized framework sequences.
 3. The antibody of claim 1, wherein theantibody is an antigen-binding fragment.
 4. The antibody of claim 1 thatbinds human ErbB3 comprising: (i) an immunoglobulin heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 2, and (ii) animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:
 4. 5. The antibody of claim 1 that binds humanErbB3 comprising: (i) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 109, and (ii) an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO:
 111. 6. The antibody ofclaim 4, wherein the antibody is a monoclonal antibody or an antigenbinding fragment thereof.
 7. The antibody of claim 1, wherein theantibody has a K_(D) of 200 pM or lower as measured by surface plasmonresonance.